hepatitis c viruses

v

ContributorsGlen N. Barber*Room 511 Papanicolau Building1550 NW 10th Ave [M710]University of Miami School of MedicineMiamiFL 33136USA

Keril J. Blight*Department of Molecular MicrobiologyCenter for Infectious Disease ResearchWashington University School of Medicine660 South Euclid AveCampus Box 8230St. LouisMO 63110-1093USA

D. Spencer CarneyDepartment of Digestive and Liver DiseasesUniversity of Texas Southwestern Medical Center5323 Harry Hines Blvd.Dallas TX75390-9048USA

Stéphane ChevaliezDepartment of VirologyINSERM U635Henri Mondor HospitalUniversity of Paris 12CréteilFrance

Linda B. CoutoBenitec LLC2375 Garcia AveMountain ViewCA 94043USA

Michael Gale Jr.*Department of MicrobiologyUniversity of Texas Southwestern Medical Center5323 Harry Hines Blvd.Dallas TX75390-9048USA

Jean Dubuisson*CNRS-UPR2511Institut de Biologie de Lille - Institut Pasteur de LilleLilleFrance

David N. Frick*Department of Biochemistry and Molecular BiologyNew York Medical CollegeValhallaNY 10595USA

Jeffrey S. Glenn*Division of Gastroenterology and HepatologyStanford University School of MedicineCCSR BuildingRoom 3115269 Campus DrivePalo AltoCA 94305-5187USA

Anne GoffardCNRS-UPR2511Institut de Biologie de Lille - Institut Pasteur de LilleLilleFrance

David R. Gretch*Laboratory MedicineVirologyBox 359690325 9th AveSeattleWA 98104-2499USA

Xiao-Song He*VA Medical Center 154CBuilding 101Room C4-1713801 Miranda Ave. Palo AltoCA94304USA

vi

Yupeng HeAbbott LaboratoriesAbbott ParkIL 60064USA

Cheng KaoDepartment of Biochemistry and BiophysicsTexas A&M UniversityCollege StationTX 77843USA

Takanobu KatoLiver Diseases BranchNIDDKNational Institute of HealthBethesdaMaryland 20892USA

Kevin C. KleinDepartment of PathobiologyUniversity of WashingtonSeattleWAUSA

Alexander A. Kolykhalov*Benitec LLC2375 Garcia AveMountain ViewCA 94043USA

Michael M. C. Lai*University of Southern CaliforniaKeck School of Medicine2011 Zonal AvenueLos AngelesCA 90033USA

Muriel LavieCNRS-UPR2511Institut de Biologie de LilleInstitut Pasteur de LilleLilleFrance

Chao Lin*Department of Infectious DiseasesVertex Pharmaceuticals Incorporated130 Waverly StreetCambridgeMassachusetts 02139USA

Jaisri R. LingappaDepartment of PathobiologyUniversity of WashingtonSeattleWAUSA

Ayaz M. MajidDepartment of Microbiology and ImmunologyUniversity of Miami School of Medicine and Sylvester Comprenhensive Cancer CenterMiamiFloridaUSA

Tatsuo MiyamuraDepartment of Virology IINational Institute of Infectious Diseases1-23-1 ToyamaShinjuku-kuTokyo 162-8640Japan

Elizabeth A. NorgardDepartment of Molecular MicrobiologyCenter for Infectious Disease ResearchWashington University School of Medicine660 South Euclid AveCampus Box 8230St. LouisMO 63110-1093USA

Arnim Pause*McGill Cancer Center and Department of BiochemistryMcGill UniversityMontrealQuebecH3G 1Y6Canada

vii

* Corresponding author

Jean-Michel Pawlotsky*Department of VirologyINSERM U635Henri Mondor HospitalUniversity of Paris 12CréteilFrance

Stephen J. Polyak*Department of Laboratory MedicineVirology DivisionBox 359690325 9th Ave.SeattleWA 98104-2499USA

C. T. Ranjith-Kumar*Department of Biochemistry and BiophysicsTexas A&M UniversityCollege StationTX 77843USA

Stephanie T. ShiDepartment of VirologyPfizer Inc.San DiegoCA 92121USA

Ikuo ShojiDepartment of Virology IINational Institute of Infectious Diseases1-23-1 ToyamaShinjuku-kuTokyo 162-8640Japan

Ella H. SklanDivision of Gastroenterology and HepatologyStanford University School of MedicineCCSR BuildingRoom 3115269 Campus DrivePalo AltoCA 94305-5187USA

Kirk A. StaschkeLilly Research LaboratoriesIndianapolisIN 46285USA

Seng-Lai Tan*Lilly Research LaboratoriesIndianapolisIN 46285USA

Takaji Wakita*Department of MicrobiologyTokyo Metropolitan Institute for Neuroscience2-6 MusashidaiFuchuTokyo 183-8526Japan

Sarah WelbournMcGill Cancer Center and Department of BiochemistryMcGill UniversityMontrealQuebecH3G 1Y6Canada

ix

Preface

Chronic hepatitis C is a serious public health problem and a disease burden in many parts of the world. The discovery of the causative agent, hepatitis C virus (HCV), in 1989 has initiated an almost unparalleled research activity in academic and pharmaceutical-industry laboratories over the ensuing years. This book aims to provide a state-of-the-art account of recent advances in the molecular and cellular biology, immunology and pathogenesis of HCV. It also aspires to discuss new strategies as well as outstanding issues for future research.

Hepatitis C has been dubbed the "silent epidemic" because it is generally asymptomatic for decades after infection; its victims often are unaware of the infection until it is too late for therapy. What is the genetic makeup and molecular features that make HCV such a "silent" yet deadly assassin? This question, in fact, is the premise by which this monograph was prepared – it was an attempt to decode the secrets of HCV, one viral gene at a time. To that end, we assembled a team of highly regarded experts from different disciplines who have prepared 16 chapters on various aspects of HCV, including the HCV genome and the role(s) of each viral gene product within the context of the viral life cycle, host interactions, and regulation of host antiviral defense and adaptive immunity.

This book can be divided into six main sections. The Introduction sets the stage by providing an overview of the history and the significant hallmarks in the discovery, diagnosis and initial treatments of HCV infection. In the first section, the authors provide an overview of our present understanding of the HCV genome, the structure and replication of these viruses (Chapter 1) and the role of the non-coding regions of HCV in regulating HCV gene expression and RNA replication (Chapter 2). The next two sections include in-depth reviews of the structural (Chapters 3 and 4) and nonstructural (Chapters 5-10) proteins of HCV. A major drawback in the past has been the lack of a robust cell-culture and small-animal model system for HCV infection and replication. However, substantial scientific progress has been made in recent years (Chapters 11-12). Armed with these tools, we are beginning to dissect the molecular mechanisms by which the virus disrupts the host innate and adaptive immune response (Chapters 13 and 14), yielding novel insights into the pathogenicity of HCV.

The final section covers the development of infectious HCV-like particle systems (Chapter 15) and the recently developed robust in vitro HCV infection systems based on the JFH-1strain (Chapter 16), which should greatly expedite our study of the full viral life cycle, and our efforts to construct anti-viral strategies and to develop effective immunization strategies with prophylactic and therapeutic

x

potential. Needless to say, this is the Holy Grail of HCV research considering that there is no vaccine available and current treatments fail in about half of HCV-infected patients.

In the meantime, biotechnology and pharmaceutical companies are making exciting progress in discovery and development of new drugs for HCV therapeutic intervention. These have been the subject of many excellent recent reviews and thus will not be covered in this book. Although it is likely to be several years before any new drug candidate will be available as an anti-HCV agent, the clinical pipeline for hepatitis C is starting to show promise for safer and more effective therapies.

Seng-Lai Tan, Ph.D.

Indianapolis, May 2006

1

Introduction

Introduction

David R. Gretch

When the term emerging infectious diseases is loosely applied, then chronic hepatitis C is recognized as one of the most important new diseases afflicting man. The term paradigm is useful when describing this disease, since the discovery, diagnosis and initial treatments of hepatitis C virus infection are all perfect examples of the increasing impact molecular biology is currently having on disease management throughout the globe. The discovery of HCV in the late 1980s occurred without the aid of conventional tissue culture or classical virological methods other than the essential reliance of the chimpanzee model for propagation and initial definition of the infectious agent as an enveloped RNA virus. Reverse transcription and PCR amplification of a subgenomic fraction of the HCV genome not only lead to the initial genetic characterization of HCV as a putative member of the Pestivirus family. It also paved the way for development of the first diagnostic test, an enzyme linked immunoassay that utilized recombinant HCV protein fragments to capture HCV antibodies from patient serum and thus provide serological evidence of infection. This critical step was a major accomplishment for molecular medicine since it provided the first opportunity to positively identify individuals with this highly prevalent yet clinically silent disease.

Even though it was well established from epidemiological studies that non-A, non-B (NANB) hepatitis was efficiently transmitted by blood transfusion, and that screening blood products for anti-hepatitis B core antibody and ALT significantly reduced the incidence of post-transfusion NANB hepatitis, development of the first generation HCV antibody screening assay had an impact far greater than many medical scientists in the field had anticipated. Results of early studies indicated that up to 10% of all units of blood transfused in the U.S. prior to the discovery of HCV had lead to transmission of the infectious agent to recipients, accounting for the vast majority of cases of post-transfusion NANB hepatitis, a fact that may have been as surprising as it was fortuitous. However, this was not the whole iceberg; world wide population-based studies revealed a global seroprevalence of well over 100 million individuals, with current estimates being frequently quoted as 170 million HCV infections today. Initial studies reported that approximately 40% of HCV infections in the U.S. were "community acquired", with no known risk factors for acquisition. Subsequent epidemiological studies have suggested that many of

Gretch

2

these cases were actually associated with the most important risk factor for HCV acquisition today, namely intravenous drug use. Such studies have also led to the identification of other previously unknown risk factors, so the term community acquired hepatitis C is no longer in vogue. Thus, cloning of a portion of the HCV RNA genome and development of an effective diagnostic test for HCV antibodies unveiled the insidious disease that is so heavily researched today; this would never have occurred without the use of molecular tools.

A second major accomplishment of molecular medicine was development and standardization of tests that efficiently detect and characterize HCV nucleic acids in patient blood. Use of the reverse transcription polymerase chain reaction (RT-PCR) assay in epidemiological studies revealed that of all patients acquiring post-transfusion hepatitis C, over 80% became chronically infected with persistent viremia for decades if not for life. Being able to define HCV viremia in a patient with a risk factor for infection or an asymptomatic individual with serological evidence of exposure to HCV has become a hallmark tool for hepatitis C management in the clinical setting from several perspectives. Confirmation of viremia equals confirmation of active disease. Since most patients with hepatitis C are asymptomatic, a large percentage has normal ALT levels in the blood, and up to 20% of infections spontaneously resolve, knowing HCV infection status is critical for defining subsequent management. Determining whether the disease is mild, moderate or severe still requires a liver biopsy unless the patient has clinical evidence of liver decompensation.

The ability to detect, quantify and genetically characterize HCV RNA in patients had an irreplaceable impact on our understanding of hepatitis C disease long before the molecular studies described in the following chapters began to unravel the complex mysteries associated with this truly unique virus-host relationship. Studies of HCV molecular epidemiology indicated that six distinct genotypes have evolved over centuries throughout the world. From clinical studies we learned that HCV persistently replicates in humans for decades, maintaining remarkably constant serum titers that often exceed 1 million viral genomes per milliliter of serum. Pharmacodynamic studies indicated that the HCV production rate exceeds one trillion new virions per day in the face of active immune responses, which is remarkable because this level of virus production is often without overt detriment to the infected host. However, HCV continuously evolves within the host as a pool of genetic variants termed viral quasispecies, presumably as an adaptation to host pressure. How host pressures shape these viral quasispecies without causing significant perturbations in HCV RNA titers is also a mystery, as is the mechanistic relationship between host pressure, viral evolution and disease progression. Again, development of HCV nucleic acid-based assays was an essential contribution of molecular medicine in terms of furthering our understanding of the fundamental

3

Introduction

virology of HCV infection in humans, and defining the mysteries of viral pathogenesis that may never be approachable for study by in vitro models.

An additional point to touch on with regards to the contribution of molecular medicine to hepatitis C disease pertains to recombinant human interferon, a drug that was engineered from the human genome many decades ago as new wonder drug for the treatment of cancer. Although the utility of interferon in treating cancer should not be understated, it was the astute observations of clinical investigators in the pre-hepatitis C era that interferon lead to normalization of serum ALT levels in about 50% of subjects treated for NANB hepatitis, a remarkable finding even before the discovery of the etiological agent. Long-term studies indicated that although many of these patients relapsed after completion of interferon treatment, several patients continue to have durable and sustained responses with long-term clearance of HCV RNA from blood. Thus, it is not unreasonable to assume that exogenous interferon alone can lead to a cure of this highly efficient virus from the infected host. We now know that HCV genotype and viral load are independent predictors of response to interferon, and other viral factors have also been implicated in influencing treatment outcome.

Molecular testing also played an essential role in the optimization of therapy for hepatitis C. Sentinel studies of HCV RNA dynamics following acute interferon dosing not only revealed a rapid dose response effect that was not previously recognized, they also lead directly to the understanding that thrice weekly dosing of interferon was not optimal. At the same time came the serendipitous discovery that the more traditional antiviral agent ribavirin potentiates long-term response to interferon by greatly reducing post-therapy relapse. The end result: the development and licensing of a much more effective combination therapy for hepatitis C, including a pegylated interferon compound with extended half-life, plus ribavirin. Today combination therapy gives clinicians the ability to achieve sustained clearance of HCV and subsequent improvements in liver disease in over 50% of their treated patients. This is an outstanding accomplishment when one considers the relatively poor prognosis for durable sustained remissions in other insidious chronic diseases in humans. Optimization of therapy through traditional clinical trial research without the use of molecular analysis of HCV RNA may never have lead to such a dramatic improvement in hepatitis C treatment outcome. It is at this point that present research takes over with the clear goal of developing new therapies capable of improving long-term response rates in those patients who remain resistant to the best available conventional therapies.

It is this problem combined with the perplexing molecular clinical biology of chronic hepatitis C that has fueled the enormous surge in basic research that is the topic of the following chapters. Over a decade of research in the chimpanzee model has

Gretch

4

provided much relevant information with respect to HCV infection and immunity in the host, and small animal models have been developed which should become important tools for further characterizing HCV biology in the near future. Aside from the ever growing body of knowledge related to basic HCV virology, several key interactions between HCV proteins and the host cell regulatory pathways have now been described, including some which have exciting potential in terms of designing new approaches to therapy. Development of the HCV replicon provided for the first time a highly efficient system for studying HCV protein function during viral replication and the effects of experimental drugs on specific aspects of the viral life cycle. However, one important limitation of the HCV replicon is the fact that infectious virus is not produced; thus it falls short of the ideal. Although the lack of a robust tissue culture system has been a major impediment to HCV research in the past, productive infection of culture cells by a unique HCV isolate has very recently been reported. It is the hope of investigators that this system will now provide the opportunity to study for the first time several essential steps in the HCV life cycle. However, it is also essential that more flexible and even more robust infection models be continuously developed.

In summary, both the intensity and breadth of HCV research are growing at a remarkable pace, and exciting new discoveries are becoming almost commonplace in the literature. The following chapters were written to provide in-depth reviews of several of the most critical areas of HCV molecular research today. However, it is the goal of this Introduction to remind readers and investigators that hepatitis C disease is highly complex and very likely involves multiple poorly defined viral-host interactions that still cannot be and may never be recapitulated in any animal model or in vitro system. For this reason, molecular research into other Pestivirus animal disease models should be pursued with renewed vigor. Finally, continuous research in the human disease model is essential for defining the most important questions for in vitro study, as is the continuous development of new molecular tools for dissecting the intriguing biopathogenesis of chronic hepatitis C in man. Just as the progress on this disease to date has been phenomenal, so too will be the future progress in furthering our understanding of HCV infection, replication, and molecular biology, and in improving the treatment of hepatitis C. The present state of progress and unanswered questions currently facing molecular investigators are both very well summarized in the following chapters. As for molecular medicine, hepatitis C may long remain the essential paradigm of how new technologies can impact in a very real manner existing problems afflicting man.

5

Genome and Life Cycle

Chapter 1

HCV Genome and Life Cycle

Stéphane Chevaliez and Jean-Michel Pawlotsky

ABSTRACTHepatitis C virus (HCV) infection afflicts more than 170 million people worldwide, with the great majority of patients with acute hepatitis C developing chronic HCV infection. It can ultimately result in liver cirrhosis, hepatic failure or hepatocellular carcinoma, which are responsible for hundreds of thousands of deaths each year. Despite the discovery of HCV over 15 years ago, our knowledge of the HCV lifecycle has been limited by our inability to grow the virus in cell culture, as well as by the lack of small-animal models of HCV infection. Nevertheless, data accumulated through the use of multiple in vitro and in vivo study systems have provided a general picture of the biology of HCV, although sometimes with contradictory results. Herein, we summarize our current understanding of the HCV genome and how its structure and encoded gene products, in a complex interplay with host cell factors, might orchestrate a productive viral lifecycle while evading the scrutiny of the host immune system. The recently developed robust in vitro HCV infection systems should help fill in some of the gaps in understanding the HCV lifecycle in the next few years.

HCV GENOME ORGANIZATION AND FUNCTIONThe Flaviviridae family is divided into three genera: flavivirus, pestivirus, and hepacivirus. Flaviviruses include yellow fever virus, dengue fever virus, Japanese encephalitis virus, and Tick-borne encephalitis virus. Pestiviruses include bovine viral diarrhea virus, classical swine fever virus and Border disease virus. HCV, with at least 6 genotypes and numerous subtypes, is a member of the hepacivirus genus, which includes tamarin virus and GB virus B (GBV-B) and is closely related to human virus GB virus C (GBV-C) (Lindenbach and Rice, 2001).

The members of the Flaviviridae family share a number of basic structural and virological characteristics. They are all enveloped in a lipid bilayer in which two or more envelope proteins (E) are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein (core or C), and contains the RNA genome. The Flaviviridae genome is a positive-strand RNA molecule ranging in size from 9.6 to 12.3 thousand nucleotides (nt), with an open reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more.

Chevaliez and Pawlotsky

6

The structural proteins are encoded by the N-terminal part of the ORF, whereas the remaining portion of the ORF codes for the nonstructural proteins (Fig. 1). Sequence motif-conserved RNA protease-helicase and RNA-dependant RNA polymerase (RdRp) are found at similar locations in the polyproteins of all of the Flaviviridae (Miller and Purcell, 1990). In addition, all Flaviviridae share similar polyprotein hydropathic profile, with flaviviruses and hepaciviruses being closer to each other than to pestiviruses (Choo et al., 1991). The ORF is flanked in 5' and 3' by untranslated regions (UTR) of 95-555 and 114-624 nt in length, respectively, which play an important role in polyprotein translation and RNA replication (Fig. 1) (Thurner et al., 2004).

Flaviviridae bind to one or more cellular receptors organized as a receptor complex and appear to trigger receptor-mediated endocytosis. Fusion of the virion envelope with cellular membranes delivers the nucleocapsid to the cytoplasm. After decapsidation, translation of the viral genome occurs in the cytoplasm, leading to the production of a precursor polyprotein, which is then cleaved by both cellular and viral proteases into structural and nonstructural proteins. Replication of the viral genome is carried out by the viral replication complex which is associated with cellular membranes and resistant to actinomycin D. Viral replication occurs in the cytoplasm via the synthesis of full-length negative-strand RNA intermediates. Progeny virions are assembled from cytoplasmic vesicles formed by budding

Fig. 1. Organization of Flaviviridae genomes. The figure shows, from top to bottom, the genomes of HCV (hepacivirus), pestivirus, and yellow fever virus (flavivirus). NS: non structural.

7


through intracellular membranes. Finally, mature virions are released into the extracellular milieu by exocytosis.

Despite the above-mentioned similarities with the members of other Flaviviridae genera, HCV does exhibit a number of virological, epidemiological as well as pathophysiological differences. Flavivirus translation is cap-dependent, i.e. mediated by a type I cap structure located in the 5'UTR (m7GpppAmp), followed by the conserved AG sequence and a relatively short stretch upstream of the polyprotein coding region (Brinton and Dispoto, 1988). In contrast, the HCV 5'UTR is not capped and, like that of pestiviruses and GB viruses, folds into a complex secondary RNA structure forming, together with a portion of the core-coding domain, an internal ribosome entry site (IRES) that mediates direct binding of ribosomal subunits and cellular factors and subsequent translation (see Chapter 2). Whereas the flavivirus 3' UTR is highly structured, the HCV 3'UTR is relatively short, less structured and contains a poly-uridyl tract that varies in length.

HCV has a narrow host specificity and tissue tropism. HCV is transmitted exclusively through direct blood-to-blood contacts between humans. Flaviviruses are principally vectored by mosquitoes or ticks and can infect a broad range of vertebrate animals, with humans being a dead-end host that does not participate in the perpetuation of virus transmission. No known pestivirus can infect humans and no known insect vector has been identified. Infections caused by flaviviruses are acute-limited in vertebrate animals, whereas HCV has a high chronicity rate in humans (50%-80%, depending on the age at infection). Strong and adapted humoral and cellular immune responses have been shown to be involved in flavivirus and pestivirus infection recovery and protection. However, HCV infection induces an immune response that fails to prevent chronicity in most cases and does not confer protection against reinfection with homologous and heterologous strains in the chimpanzee model (Farci et al., 1997).

HCV GENOME STRUCTURE AND ORGANIZATIONThe structural organization of HCV genome is schematically depicted in Fig. 2.

5' UNTRANSLATED REGIONThe HCV 5'UTR contains 341 nt located upstream of the ORF translation initiation codon. It is the most conserved region of the genome (nt sequence identity is 60% with GBV-B and approximately 50% with pestiviruses (Choo et al., 1991; Han et al., 1991). The 5'UTR contains four highly structured domains, numbered I to IV, containing numerous stem-loops and a pseudoknot (Brown et al., 1992; Wang et al., 1995). Domains II, III and IV together with the first 12 to 30 nt of the core-coding region constitute the IRES (Honda et al., 1996). Structural characterization by electron microscopy (EM) indicated that domains II, III and IV form distinct


8

regions within the molecule, with a fl exible hinge between domains II and III (Beales et al., 2001). The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors, an event that likely constitutes the fi rst step of HCV polyprotein translation.

Several reports suggested a tissue compartmentalization of IRES sequences (Laskus et al., 2000; Lerat et al., 2000; Nakajima et al., 1996; Shimizu et al., 1997). Infection of lymphoid cell lines with HCV genotype 1a H77 strain led to the selection of a quasispecies with nucleotide substitutions within the 5'UTR relative to the inoculum that conferred a 2- to 2.5-fold increase in translation effi ciency in human lymphoid cell lines relative to monocyte, granulocyte or monocyte cell lines (Lerat et al., 2000). Furthermore, different translation effi ciencies of HCV quasispecies variants isolated from different cell types in the same patient were observed, suggesting cell type-specifi c IRES interactions with cellular factors may also modulate polyprotein translation (Forton et al., 2004; Laporte et al., 2000; Lerat et al., 2000).

Fig. 2. HCV genome organization (top) and polyprotein processing (bottom). The 5'UTR consists of four highly structured domains and contains the IRES. The 3'UTR consists of stable stem-loop structures and an internal poly(U)-poly(U/C) tract. The central 9.6-kb ORF codes for a polyprotein of slightly more than 3000 aa depending on the HCV genotype. S and NS correspond to regions coding for structural and nonstructural proteins, respectively. The polyprotein processing and the location of the 10 HCV proteins relative to the ER membrane are schematically represented. Scissors indicate ER signal peptidase cleavage sites; cyclic arrow, autocatalytic cleavage of the NS2-NS3 junction; black arrows, NS3-NS4A protease complex cleavage sites; intramembranous arrow, cleavage by the signal peptide peptidase. The transmembrane domains of E1 and E2 are shown after signal-peptidase cleavage and reorientation of the respective C-terminus hydrophobic stretches (dotted rectangles). Spots denote glycosylation sites of the E1 and E2 envelope proteins. Reproduced from Penin et al., 2004b with permission.

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3' UNTRANLATED REGIONThe 3'UTR contains approximately 225 nt. It is organized in three regions including, from 5' to 3', a variable region of approximately 30-40 nt, a long poly(U)-poly(U/UC) tract, and a highly conserved 3'-terminal stretch of 98 nt (3'X region) that includes three stem-loop structures SL1, SL2 and SL3 (Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996). The 3'UTR interacts with the NS5B RdRp and with two of the four stable stem-loop structures located at the 3' end of the NS5B-coding sequence (Cheng et al., 1999; Lee et al., 2004). The 3'X region and the 52 upstream nt of the poly(U/C) tract were found to be essential for RNA replication, whereas the remaining sequence of the 3'UTR appears to enhance viral replication (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b).

CHARACTERISTICS AND FUNCTIONS OF HCV PROTEINSThe HCV ORF contains 9024 to 9111 nt depending on the genotype. The ORF encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and E2), a small protein, p7, whose function has not yet been definitively defined, 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), and the so-called "F" protein which results from a frameshift in the core coding region (Fig. 2; Table 1). The characteristics and functions of HCV proteins are extensively

Table 1. HCV proteins and their functions in the viral life cycle. Adapted from Bartenschlager et al., 2004.HCV protein Function Apparent molecular weight

(kDa)Core Nucleocapsid 23 (precursor)

21 (mature)F/ARFa-protein ? 16-17E1 Envelope

Fusion domain? 33-35

E2 EnvelopeReceptor bindingFusion domain?

70-72

p7 Calcium ion channel (viroporin) 7NS2 NS2-3 autoprotease 21-23NS3 Component of NS2-3 and NS3-4A proteinases

NTPase/helicase69

NS4A NS3-4A proteinase cofactor 6NS4B Membranous web induction 27NS5A RNA replication by formation of replication

complexes56 (basal form)

58 (hyperphosphorylatedform)

NS5B RNA-dependant RNA polymerase 68a Frameshift/ alternate reading frame


10

described elsewhere in this book. Here, we provide a brief overview of the viral gene products and their roles in the HCV lifecycle.

STRUCTURAL PROTEINS

CORE PROTEIN

The HCV core protein is a highly basic, RNA-binding protein, which presumably forms the viral capsid (see Chapter 3). The HCV core protein is released as a 191 aa precursor of 23-kDa (P23). Although proteins of various sizes (17 to 23 kDa) were detectable, the 21-kDa core protein (P21) appeared to be the predominant form (Yasui et al., 1998). The core protein contains three distinct predicted domains : an N-terminal hydrophilic domain of 120 aa (domain D1), a C-terminal hydrophobic domain of about 50 aa (domain D2), and the last 20 or so aa that serve as a signal peptide for the downstream envelope protein E1 (Grakoui et al., 1993c; Harada et al., 1991; Santolini et al., 1994). Domain D1 contains numerous positive charges. It is principally involved in RNA binding and nuclear localization, as suggested by the presence of three predicted nuclear localization signals (NLS) (Chang et al., 1994; Suzuki et al., 1995; Suzuki et al., 2005). Domain D2 is responsible for core protein association with endoplasmic reticulum (ER) membranes, outer mitochondria membranes and lipid droplets (Schwer et al., 2004; Suzuki et al., 2005).

Both membrane-bound and membrane-free core proteins appear to exist as dimeric or multimeric forms. When expressed in various in vitro systems, including cell-free or mammalian, bacterial, insect or yeast cell culture models, the HCV core protein can form nucleocapsid-like particles (NLPs) (Baumert et al., 1998; Blanchard et al., 2002; Blanchard et al., 2003; Dash et al., 1997; Ezelle et al., 2002; Iacovacci et al., 1997; Klein et al., 2004; Mizuno et al., 1995; Pietschmann et al., 2002; Serafino et al., 1997; Shimizu et al., 1996). The region between aa 82 and 102 of hydrophilic domain D1 contains a tryptophan-rich sequence and has been suggested to allow the P21 core protein to interact with itself, a property not borne by the precursor P23 (Nolandt et al., 1997). On the other hand, the 75 N-terminal residues of the core protein appear sufficient for NLP assembly in a bacterial system (Majeau et al., 2004). Recently, two clusters of basic residues located in the 68 N-terminal nt were shown to play a critical role in capsid assembly in a cell-free system, whereas the region between aa 82 and 102 did not play a major role (Klein et al., 2005). The critical residues for capsid assembly remain to be precisely identified.

In addition to its role in viral capsid formation, the core protein has been suggested to directly interact with a number of cellular proteins and pathways that may be important in the viral lifecycle (McLauchlan, 2000). The HCV core protein has pro- and anti-apoptotic functions (Chou et al., 2005; Kountouras et al., 2003; Meyer et al., 2005), stimulates hepatocyte growth in Huh-7 cell line by transcriptional upregulation of growth-related genes (Fukutomi et al., 2005), and has been

11


implicated in tissue injury and fibrosis progression (Nunez et al., 2004). The HCV core protein could also regulate the activity of cellular genes, including c-myc and c-fos, and alter the transcription of other viral promoters (Ray et al., 1995; Shih et al., 1993). It induces hepatocellular carcinoma when expressed in transgenic mice (Moriya et al., 1998; Moriya et al., 1997). It could also induce the formation of lipid droplets and may play a direct role in steatosis formation (Barba et al., 1997; Moriya et al., 1998; Moriya et al., 1997).

E1 AND E2 ENVELOPE GLYCOPROTEINS

The two envelope glycoproteins, E1 and E2, are essential components of the HCV virion envelope and necessary for viral entry and fusion (Bartosch et al., 2003a; Nielsen et al., 2004) (see Chapter 4). E1 and E2 have molecular weights of 33-35 and 70-72 kDa, respectively, and assemble as noncovalent heterodimers (Deleersnyder et al., 1997). E1 and E2 are type I transmembrane glycoproteins, with N-terminal ectodomains of 160 and 334 aa, respectively, and a short C-terminal transmembrane domain of approximately 30 aa. The E1 and E2 transmembrane domains are composed of two stretches of hydrophobic aa separated by a short polar region containing fully conserved charged residues. They have numerous functions, including membrane anchoring, ER localization and heterodimer assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). The ectodomains of E1 and E2 contain numerous proline and cysteine residues, but intramolecular disulfide bonds have not been observed (Matsuura et al., 1994). E1 and E2 are highly glycosylated, containing up to 5 and 11 glycosylation sites, respectively. In addition, E2 contains hypervariable regions with aa sequences differing up to 80% between HCV genotypes and between subtypes of the same genotype (Weiner et al., 1991). Hypervariable region 1 (HVR1) contains 27 aa and is a major (but not the only) HCV neutralizing epitope (Farci et al., 1996; Zibert et al., 1997). Despite the HVR1 sequence variability, the physicochemical properties of the residues at each position and the overall conformation of HVR1 are highly conserved among all known HCV genotypes, suggesting an important role in the virus lifecycle (Penin et al., 2001).

E2 plays a crucial role in the early steps of infection. Viral attachment is thought to be initiated via E2 interaction with one or several components of the receptor complex (Flint and McKeating, 2000; Rosa et al., 1996). Because HVR1 is a basic region with positively charged residues located at specific sequence positions, it can theoretically interact with negatively charged molecules at the cell surface. This interaction could play a role in host cell recognition and attachment, as well as in cell or tissue compartmentalization (Barth et al., 2003; Bartosch et al., 2003b). In addition, it was recently shown that human serum facilitated infection of Huh7 cells by HCV pseudoparticles, apparently mediated through an interplay between serum high-density lipoproteins (HDL), HVR1 and the scavenger receptor B type I (SR-BI) (Bartosch et al., 2005; Voisset et al., 2005). Less is known about E1, but


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it is thought to be involved in intra-cytoplasmic virus-membrane fusion (Flint and McKeating, 2000; Rosa et al., 1996).

FRAMESHIFT PROTEINThe F (frameshift) protein or ARFP (alternate reading frame protein) is generated as a result of a -2/+1 ribosomal frameshift in the N-terminal core-encoding region of the HCV polyprotein. Antibodies to peptides from the F protein were detected in chronically infected patients, suggesting that the protein is produced during infection (Walewski et al., 2001). However, the exact translational mechanisms governing the frequency and yield of the F protein during the various phases of HCV infection are completely unknown. Thus, the role of F protein in the HCV lifecycle remains enigmatic but it was proposed to be involved in viral persistence (Baril and Brakier-Gingras, 2005).

NONSTRUCTURAL PROTEINS

P7

p7 is a small, 63 aa polypeptide, that has been shown to be an integral membrane protein (Carrere-Kremer et al., 2002). It comprises two transmembrane domains organized in α-helices, connected by a cytoplasmic loop. p7 appears to be essential, because mutations or deletions in its cytoplasmic loop suppressed infectivity of intra-liver transfection of HCV cDNA in chimpanzees (Sakai et al., 2003). In vitro studies suggested that p7 belongs to the viroporin family and could act as a calcium ion channel (Gonzalez and Carrasco, 2003). However, these results remain to be confirmed in vivo.

NS2

NS2 is a non-glycosylated transmembrane protein of 21-23 kDa (see Chapter 5). It contains two internal signal sequences at aa positions 839-883 and 928-960, which are responsible for ER membrane association (Santolini et al., 1995; Yamaga and Ou, 2002). NS2, together with the amino-terminal domain of the NS3 protein, the NS2-3 protease, constitutes a zinc-dependent metalloprotease that cleaves the site between NS2 and NS3 (Grakoui et al., 1993b; Grakoui et al., 1993c; Hijikata et al., 1993). NS2 is a short-lived protein that looses its protease activity after self-cleavage from NS3 and is degraded by the proteasome in a phosphorylation-dependent manner by means of protein kinase casein kinase 2 (Franck et al., 2005). In addition to its protease activity, NS2 could interact with host cell proteins, such as the liver-specific pro-apoptotic cell death-inducing DFF45-like effector (CIDE-B), and affect reporter genes controlled by liver and non-liver-specific promoters and enhancers (Dumoulin et al., 2003; Erdtmann et al., 2003). However, the consequences of such interactions within the context of the HCV lifecyle are not clear.

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NS3-NS4A

NS3 is a multi-functional viral protein containing a serine protease domain in its N-terminal third and a helicase/NTPase domain in its C-terminal two-thirds. NS4A is a cofactor of NS3 protease activity. NS3-4A also bears additional properties through its interaction with host cell pathways and proteins that may be important in the lifecycle and pathogenesis of infection (see Chapters 6 and 13). Not surprisingly, the NS3-NS4A protease is one of the most popular viral targets for anti-HCV therapeutics (Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

NS3-NS4A PROTEASE

The NS3-NS4A protease is essential for the HCV lifecycle. It catalyzes HCV polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. The 3D structure of the NS3 serine protease domain complexed with NS4A has been determined (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). The catalytic triad is formed by residues His 57, Asp 81 and Ser 139 (Bartenschlager et al., 1993; Grakoui et al., 1993a; Tomei et al., 1993). The central region of NS4A (aa 21–30) acts as a cofactor of NS3 serine protease activity, allowing its stabilization, localization at the ER membrane as well as cleaveage-dependent activation, particularly at the NS4B/NS5A junction (Bartenschlager et al., 1995; Lin et al., 1995; Tanji et al., 1995).

Recently, HCV NS3-NS4A was shown in vitro to antagonize the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathway, an important mediator of interferon induction in response to a viral infection (Foy et al., 2003). NS3-NS4A also appears to prevent dsRNA signaling via the toll-like receptor 3 upstream of IRF-3 (Li et al., 2005). One potential mechanism includes a blockade of the intracellular double-stranded RNA sensor protein (RIG-I) pathway by NS3-NS4A (Sumpter et al., 2005). Thus, HCV could utilize NS3-4A protease to circumvent the innate immune response at the early stages of infection. In addition, NS3 was also reported to induce malignant transformation of NIH3T3 cells (Sakamuro et al., 1995), suppress actinomycin D-induced apoptosis in murine cell lines (Fujita et al., 1996), and to be involved in hepatocarcinogenesis events (Borowski et al., 1996; Hassan et al., 2005), although the exact mechanisms are not clear.

NS3 HELICASE-NTPASE

The NS3 helicase-NTPase domain consisting of the 442 C-terminal aa of the NS3 protein is a member of the helicase superfamily-2 (see Chapter 7). Its three-dimentional structure has also been determined (Cho et al., 1998; Kim et al., 1998; Yao et al., 1997). The NS3 helicase-NTPase has several functions, including RNA-stimulated NTPase activity, RNA binding, and unwinding of RNA regions of extensive secondary structure by coupling unwinding and NTP hydrolysis (Gwack et al., 1997; Tai et al., 1996). During RNA replication, the NS3 helicase has been suggested to translocate along the nucleic acid substrate by changing protein


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conformation, utilizing the energy of NTP hydrolysis. A recent study proposed that the helicase directional movement step is fueled by single-stranded RNA binding energy, while NTP binding allows for a brief period of random movement that prepares the helicase for the next cycle (Levin et al., 2005). In addition, NS3 helicase activity appears to be modulated by the NS3 protease domain and the NS5B RdRp (Zhang et al., 2005).

NS4B

NS4B is an integral membrane protein of 261 aa with an ER or ER-derived membrane localization (Hugle et al., 2001; Lundin et al., 2003). NS4B is predicted to harbor at least four transmembrane domains and an N-terminal amphipathic helix that are responsible for membrane association (Elazar et al., 2004; Hugle et al., 2001; Lundin et al., 2003). One of the functions of NS4B is to serve as a membrane anchor for the replication complex (see Chapter 8) (Egger et al., 2002; Elazar et al., 2004; Gretton et al., 2005). Additional putative properties include inhibition of cellular syntheses (Florese et al., 2002; Kato et al., 2002), modulation of HCV NS5B RdRp activity (Piccininni et al., 2002), transformation of NIH3T3 cell lines (Park et al., 2000), and induction of interleukin 8 (Kadoya et al., 2005).

NS5A

NS5A is a 56-58 kDa phosphorylated zinc-metalloprotein that probably plays an important role in virus replication and regulation of cellular pathways (see Chapter 9). The N-terminal region of NS5A (aa 1-30) contains an amphipathic α-helix that is necessary and sufficient for membrane localization in perinuclear membranes as well as for assembly of the replication complex (Brass et al., 2002; Elazar et al., 2003; Penin et al., 2004a). Downstream of this motif, the NS5A protein was predicted to contain three domains, numbered I to III. Domain I, located at the N-terminus, contains an unconventional zinc-binding motif formed by four cysteine residues conserved among the hepacivirus and pestivirus genera (Tellinghuisen et al., 2004). HCV replicon RNA replication was inhibited by mutations in the NS5A sequence (Elazar et al., 2003; Penin et al., 2004b) and abolished by alterations of the zinc-binding site (Tellinghuisen et al., 2004). The recently determined 3-D structure of Domain I suggested the presence of protein, RNA and membrane interaction sites (Moradpour et al., 2005; Tellinghuisen et al., 2005).

The mechanisms by which NS5A regulate HCV replication are not entirely clear. NS5A associates with lipid rafts derived from intracellular membranes through its binding to the C-terminal region of a vesicle-associated membrane-associated protein of 33 kDa (hVAP-33) (Shi et al., 2003; Tu et al., 1999). This interaction appears to be crucial for the formation of the HCV replication complex in connection with lipid rafts (Gao et al., 2004). A recent study in the replicon system proposed a model in which NS5A hyperphosphorylation disrupts the interaction with hVAP-33 and negatively regulates viral RNA replication (Evans et al., 2004). Another

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report suggested that the level of NS5A phosphorylation plays an important role in the viral lifecycle by regulating a switch from replication to assembly, whereby hyperphosphorylated forms function to maintain the replication complex in an assembly-incompetent state (Appel et al., 2005). Furthermore, NS5A can interact directly with NS5B, but the mechanism by which NS5A modulates the RdRp activity has not been elucidated (Shimakami et al., 2004). In addition, NS5A was reported to interact with a geranylgeranylated cellular protein (Wang et al., 2005a). This is potentially significant considering that assembly of the viral replication complex has been shown to require geranylgeranylation of one or more host cell proteins (Ye et al., 2003).

Multiple functions have been assigned to NS5A based on its interactions with cellular proteins (Tellinghuisen and Rice, 2002) (see Chapter 9). For instance, NS5A appears to play a role in interferon resistance by binding to and inhibiting PKR, an antiviral effector of interferon-α (Gale et al., 1998). NS5A also bears transcriptional activation functions (Pellerin et al., 2004; Polyak et al., 2001) and appears to be involved in the regulation of cell growth and cellular signaling pathways (Tan and Katze, 2001; Tellinghuisen and Rice, 2002). However, these observations remain to be confirmed in vivo.

NS5B RNA-DEPENDENT RNA POLYMERASE

NS5B belongs to a class of membrane proteins termed tail-anchored proteins (Ivashkina et al., 2002; Schmidt-Mende et al., 2001) (see Chapter 10). Its C-terminal region (21 residues) forms an α-helical transmembrane domain responsible for post-translational targeting to the cytosolic side of the ER, where the functional protein domain is exposed (Moradpour et al., 2004; Schmidt-Mende et al., 2001). The crystal structure of NS5B revealed that the RdRp has a classical "fingers, palm and thumb" structure formed by its 530 N-terminal aa (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). Interactions between the fingers and thumb subdomains result in a completely encircled catalytic site that ensures synthesis of positive- and negative-strand HCV RNAs (Lesburg et al., 1999). The RdRp is another important target for the development of anti-HCV drugs (Di Marco et al., 2005; Ma et al., 2005; Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

Interactions between NS5B and cellular components have also been reported. The C-terminus of NS5B can interact with the N-terminus of hVAP-33, and the interaction may play an important role in the formation of the HCV replication complex (Gao et al., 2004; Schmidt-Mende et al., 2001). More recently, NS5B was reported to bind cyclophilin B, a cellular peptidyl-prolyl cis-trans isomerase that apparently regulates HCV replication through modulation of the RNA binding capacity of NS5B (Watashi et al., 2005).


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THE HCV LIFECYCLE

CELLULAR ATTACHMENT OF HCV VIRIONS AND ENTRYMany efforts have been made to develop models to identify candidate HCV receptors and study viral binding and entry into target cells. Various cellular and in vivo systems utilizing infected blood samples, virus-like particles produced by expression of structural HCV proteins in insect or mammalian cells, liposomes containing E1-E2, as well as pseudotype particles have yielded a considerable amount of data, although they are not always easy to reconcile. Fig. 3 summarizes the hypothetical HCV lifestyle.

HCV RECEPTORSSeveral cell surface molecules have been proposed to mediate HCV binding or HCV binding and internalization.

CD81

Among all putative HCV receptor molecules, CD81 has been the most extensively studied (Pileri et al., 1998). Human CD81 (target of antiproliferative antibody 1, TAPA-1) is a 25-kDa molecule belonging to the tetraspanin or transmembrane 4 superfamily. It is found at the surface of numerous cell types, where it is thought to assemble as homo- and/or heterodimers by means of a conserved hydrophobic interface. CD81 contains four hydrophobic transmembrane regions (TM1 to TM4) and two extracellular loop domains of 28 and 80 aa, respectively: the small extracellular loop (SEL) and the large extracellular loop (LEL). The LEL is located between TM3 and TM4. It is composed of five α-helices and contains four cysteine residues (Kitadokoro et al., 2001). The SEL is needed for optimal surface expression of the LEL (Masciopinto et al., 2001). The intracellular and transmembrane domains of CD81 are highly conserved among different species. In contrast, the LEL is variable, except between humans and chimpanzees, the only two species permissive to HCV infection (Major et al., 2004; Walker, 1997). The CD81 LEL has been shown to mediate binding of HCV through its envelope glycoprotein E2 (Pileri et al., 1998). The integrity of two disulfide bridges is necessary for the CD81-HCV interaction to occur (Petracca et al., 2000), and the site of interaction appears to involve CD81 residues 163, 186, 188 and 196 (Flint et al., 1999; Meola et al., 2000). The E2 domains involved in CD81 binding remain controversial. Early studies suggested the involvement of aa 480-493 and 544-551 in the truncated soluble form of E2 (Flint et al., 1999), whereas a more recent study pointed to a role for two other domains, including aa 613-618 and a second domain spanning the two HVRs (aa 384-410 and 476-480) (Roccasecca et al., 2003).

Several studies argue that cellular factors other than CD81 are required for HCV infection. The expression of human CD81 in a CD81-deficient human hepatoma cell line restored permissiveness to infection with HCV pseudo-particles, but a

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murine fibroblast cell line expressing human CD81 remained resistant to HCV entry (Cormier et al., 2004). In addition, expression of human CD81 in transgenic mice did not confer susceptibility to HCV infection (Masciopinto et al., 2002). It is possible that the CD81 molecule could act as a post-attachment entry co-receptor and that other cellular factors act together with CD81 to mediate HCV binding and entry into hepatocytes (Cormier et al., 2004).

SR-BI

The scavenger receptor B type I (SR-BI) has been proposed as another candidate receptor for HCV (Scarselli et al., 2002). SR-BI is a 509-aa glycoprotein with a large extracellular loop anchored to the plasma membrane at both N- and C-termini by means of transmembrane domains with short cytoplasmic extensions (Krieger, 2001). SR-BI is a fatty acylated protein located in lipid raft domains. It is expressed at high levels in hepatocytes and steroidogenic cells (Babitt et al., 1997; Krieger, 2001). The natural ligand of SR-BI is high density lipoproteins (HDL). HDLs are internalized through a non-clathrin-dependent endocytosis process that mediates cholesterol uptake and recycling of HDL apoprotein (Silver et al., 2001). HCV genotypes 1a and 1b recombinant E2 envelope glycoproteins were shown to bind HepG2 cells (a human hepatoma cell line that does not express CD81) by interacting with an 82 kDa glycosylated SR-BI molecule (Scarselli et al., 2002). Binding appeared to be highly specific: tranfection of rodent cells with human or tupaia SR-BI (88 % aa identity with human SR-BI) resulted in E2 binding, whereas neither mouse SR-BI (80 % aa identity) nor the closely related human scavenger receptor CD36 (60 % aa identity) bound E2. The SR-BI LEL appeared to be responsible for HCV binding, and HVR1 was recently suggested to be the E2 envelope region involved in the interaction, which was facilitated by serum HDLs (Bartosch et al., 2003b; Scarselli et al., 2002; Voisset et al., 2005). However, the fact that antibodies directed against SR-BI resulted only in a partial blockade of binding suggests that SR-BI is not the only cell surface molecule involved in HCV binding to hepatocytes (Barth et al., 2005).

DC-SIGN AND L-SIGN

The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN or CD209) and the liver/lymph node-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing integrin (L-SIGN or CD209L) have been proposed as tissue-specific capture receptors for HCV present in various cell types that could play a critical role in viral pathogenesis and tissue tropism (Gardner et al., 2003; Lozach et al., 2004; Lozach et al., 2003; Pohlmann et al., 2003). DC-SIGN is a 44-kDa homotetrameric type II integral membrane protein with a short amino-terminal cytoplasmic domain and a carboxy-terminal C-type (calcium-dependent) lectin domain. DC-SIGN is expressed at a high level on myeloid-lineage dendritic cells. Its interaction with ICAM-3 activates T cells (Geijtenbeek et al., 2000). L-SIGN is abundantly expressed at the surface of endothelial cells of the liver


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and lymph nodes and shares 77% aa sequence identity with DC-SIGN (Bashirova et al., 2001). A rapid internalization of virus-like particles upon capture of HCV pseudo-particles by both DC-SIGN and L-SIGN, presumably via E2 binding, was reported (Ludwig et al., 2004), although this was not observed in another study (Lozach et al., 2004).

LDL-R

The low-density lipoprotein (LDL) receptor (LDL-R) is an endocytic receptor that transports lipoproteins, mainly the cholesterol-rich LDLs, into cells through receptor-mediated endocytosis (Chung and Wasan, 2004). Virus-like particles complexed with LDLs have been reported to enter into cells via the LDL receptor (Agnello et al., 1999; Monazahian et al., 1999). In support of this view, binding of low-density HCV particles recovered from plasma by sucrose gradient sedimentation correlated with the density of LDL receptors at the surface of MOLT-4 cells and fibroblasts, and the binding was inhibited by LDL but not by soluble CD81 (Wunschmann et al., 2000).

ASIALOGLYCOPROTEIN RECEPTOR

The asialoglycoprotein receptor (ASGP-R) has been reported to mediate binding and internalization of structural HCV proteins (C-E1-E2±p7) expressed in a baculovirus system. Cotransfection of a non-permissive mouse fibroblast cell line with cDNAs of both ASGP-R subunits (H1 and H2) restored permissiveness (Saunier et al., 2003).

GLYCOSAMINOGLYCANS

Conservation of positively charged residues in the N-terminus of E2 is in keeping with a possible interaction with heparan sulfate proteoglycans (HSPG) (Barth et al., 2003). E2, in particular its HVR-1, has been shown to bind HSPG with a stronger affinity than other viral envelope glycoproteins, such as human herpes virus 8 or dengue virus envelope proteins. However, glycosaminoglycans are ubiquitously expressed as cell surface molecules. It is conceivable that HSPG could serve as the initial docking site for HCV attachment and the virus is subsequently transferred to another high-affinity receptor (or receptor complex) triggering entry (Barth et al., 2003).

MECHANISMS OF CELL ENTRY AND FUSIONAfter attachment, the nucleocapsid of enveloped viruses is released into the cell cytoplasm as a result of a fusion process between viral and cellular membranes. Fusion is mediated by specialized viral proteins and takes place either directly at the plasma membrane or following internalization of the particle into endosomes. The entry process is controlled by viral surface glycoproteins that trigger the changes required for mediating fusion. At least two different classes of fusion proteins (I and II) can be distinguished (Lescar et al., 2001). The flaviviruses enter target cells

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by receptor-mediated endocytosis and use class II fusion proteins (Lindenbach and Rice, 2001). By analogy, HCV envelope glycoproteins are believed to belong to class II fusion proteins (Yagnik et al., 2000). However, in contrast with other class II fusion proteins, HCV envelope glycoproteins do not appear to require cellular protease cleavage during their transport through the secretory pathway (Op De Beeck et al., 2004). HCV entry into cells is pH-dependent and endocytosis-dependent (Agnello et al., 1999; Bartosch et al., 2003b; Hsu et al., 2003), but the identity of the HCV fusion peptide remains controversial. E1 appeared as a good candidate because sequence analysis suggested the presence of a fusion peptide in its ectodomain (Flint and McKeating, 2000; Rosa et al., 1996). Nevertheless, E2 was shown to share structural homology with class II fusion proteins (Lescar et al., 2001; Yagnik et al., 2000). Crystallographic 3D structure determination and cryo-EM-based studies of both envelope glycoproteins are needed to better understand the mechanisms of HCV fusion.

RNA TRANSLATION AND POST-TRANSLATIONAL PROCESSING

POLYPROTEIN SYNTHESIS

Decapsidation of viral nucleocapsids liberates free positive-strand genomic RNAs into the cell cytoplasm, where they serve, together with newly synthesized RNAs, as messenger RNAs for synthesis of the HCV polyprotein. HCV genome translation is under the control of the IRES, spanning domains II to IV of the 5'UTR and the first nucleotides of the core-coding region. IRES domain I is not part of the IRES but plays an important role by modulating IRES-dependent translation (Friebe et al., 2001; Luo et al., 2003). The IRES mediates cap-independent internal initiation of HCV polyprotein translation by recruiting both cellular proteins, including eukaryotic initiation factors (eIF) 2 and 3 and viral proteins (Ji et al., 2004; Lukavsky et al., 2000; Otto and Puglisi, 2004). Three distinct translation initiation complexes (40S, 48S and 80S) are generated, as shown by in vitro translation experiments in HeLa S10 cells and rabbit reticulocyte lysates and by ex vivo experiments in mammalian cells (Kong and Sarnow, 2002).

The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors (Otto et al., 2002; Spahn et al., 2001). The 40S subunit assembles with eIF3 and this ternary complex joins with eIF2, GTP, and the initiator tRNA to form a 48S particle in which the tRNA is positioned in the P site of the 40S subunit, base-paired to the start codon of the mRNA. Upon hydrolysis of GTP, eIF2 releases the initiator tRNA and dissociates from the complex. A second GTP hydrolysis step involving initiation factor eIF5B then enables the 60S ribosomal subunit to associate, forming a functional 80S ribosome that initiates viral protein synthesis (Ji et al., 2004; Kieft et al., 2001; Otto and Puglisi, 2004; Sizova et al., 1998).


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N

M

ER

Fig. 3. Hypothetical HCV replication cycle. HCV particles bind to the host cells via a specific interaction between the HCV envelope glycoproteins and a yet unknown cellular receptor. Bound particles are probably internalized by receptor-mediated endocytosis. After the viral genome is liberated from the nucleocapsid (uncoating) and translated at the rough ER, NS4B (perhaps in conjunction with other viral or cellular factors) induces the formation of membranous vesicles (referred to as the membranous web; EM in the lower right). These membranes are supposed to serve as scaffolds for the viral replication complex. After genome amplification and HCV protein expression, progeny virions are assembled. The site of virus particle formation has not yet been identified. It may take

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A number of cellular proteins were reported to interact with the 5'UTR including the polypyrimidine tract-binding protein (PTB) (Ali and Siddiqui, 1995), heterogeneous nuclear ribonucleoprotein L (hnRNP L) (Hahm et al., 1998), La autoantigen (Ali and Siddiqui, 1997), the poly(rC)-binding protein 2 (PCP2) (Spangberg and Schwartz, 1999) and NS1-associated protein 1 (NSAP1) (Kim et al., 2004). The biological significance of these protein-RNA interactions remains unknown. In addition, HCV proteins may affect IRES translational efficiency, including the core protein (Zhang et al., 2002) and non-structural proteins NS4A and NS5B (Kato et al., 2002). The HCV 3'UTR may also modulate IRES-dependent translation, but this remains controversial (Imbert et al., 2003; Wang et al., 2005b).

POST-TRANSLATIONAL PROCESSING

HCV genome translation generates a large precursor polyprotein, which is targeted to the ER membrane for translocation of the E1 ectodomain into the ER lumen, a process mediated by the internal signal sequence located between the core and E1 sequences. Cleavage of the signal sequence by the host signal peptidase yields the immature form of the core protein (P23) (McLauchlan et al., 2002). The signal peptide is further processed by a host signal peptide peptidase (SPP, a presenilin-type aspartic protease that resides in the ER membrane) to yield the mature form of the core protein (P21) (Fig. 3) (Penin et al., 2004b). The host signal peptidase also ensures cleavage at the E1-E2 junction in the ER lumen. Additional signal peptidase cleavages at the C-terminal end of E2 and between p7 and NS2 give rise to p7 (Fig. 3). An incomplete cleavage may lead to the production of non-cleaved E2-p7 proteins, the role of which is unknown. E1 and E2 subsequently undergo several maturation steps, including N-glycosylation, conformation and assembly of E1E2 heterodimers (Penin et al., 2004b). Heterogeneous E1E2 aggregates are also produced, but their role in viral particle formation is not known.

The zinc-dependent NS2-3 auto-protease ensures cis-cleavage of NS3 from NS2 (Fig. 2). NS3 needs to assemble with its cofactor NS4A to catalyze cis-cleavage at the NS3-NS4A junction and trans-cleavage at all downstream junctions including NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B (Fig. 2) (Bartenschlager and Lohmann, 2000; Lindenbach and Rice, 2005). The cleavage sites recognized by the

place at intracellular membranes derived from the ER or the Golgi compartment. Newly produced virus particles may leave the host cell by the constitutive secretory pathway. The upper right panel of the figure shows a schematic representation of an HCV particle. The middle panel shows a model for the synthesis of negative-stranded (-) and positive stranded (+) progeny RNA via a double-stranded replicative form (RF) and a replicative intermediate (RI). The bottom panel shows an electron micrograph of a membranous web (arrow heads) in Huh7 cell containing subgenomic HCV replicons. The web is composed of small vesicles embedded in a membrane matrix. Bar: 500 nm; N: nucleus; ER: endoplasmic reticulum; M: mitochondria. Reproduced from Bartenschlager et al., 2004 with permission.


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NS3-NS4A protease have in common the following sequence: Asp/GluXXXXCys/Thr-Ser/Ala, with trans cleavages occurring downstream of a cysteine residue and the cis cleavage occurring downstream of a threonine residue.

HCV REPLICATION

THE HCV REPLICATION COMPLEX

Infection with a positive-strand RNA virus leads to rearrangements of intracellular membranes, a prerequisite to the formation of a replication complex that associates viral proteins, cellular components and nascent RNA strands. The HCV NS4B protein seems to be sufficient to induce the formation of a membranous web or membrane-associated foci (Egger et al., 2002; Gretton et al., 2005). It is not known whether NS4B recruits cellular proteins responsible for vesicle formation or induces vesicle formation by itself. The membranous web is derived from ER membranes (Bartenschlager et al., 2004). It is rich in cholesterol and fatty acids, the degree of saturation of which (that influences membrane fluidity) modulates HCV replication (Kapadia and Chisari, 2005). HCV replication was shown to occur in detergent-resistant membranes that co-localize with caveolin-2, an essential component of lipid raft domains (Shi et al., 2003). Indeed, lipid rafts are involved in the formation of the replication complex, through protein-protein interactions between hVAP-33 and both NS5A and NS5B HCV proteins (Gao et al., 2004; Shi et al., 2003; Tu et al., 1999). Overall, the membranous web consists of small vesicles embedded in a membranous matrix, forming a membrane-associated multiprotein complex that contains all of the nonstructural HCV proteins (Egger et al., 2002).

MECHANISM OF HCV REPLICATION

The precise mechanisms of HCV replication are still poorly understood. By analogy with other positive-strand RNA viruses, HCV replication is thought to be semi-conservative and asymmetric with two steps, both of which are catalyzed by the NS5B RdRp. The positive-strand genome RNA serves as a template for the synthesis of a negative-strand intermediate of replication during the first step. In the second step, negative-strand RNA serves as a template to produce numerous strands of positive polarity that will subsequently be used for polyprotein translation, synthesis of new intermediates of replication or packaging into new virus particles (Bartenschlager et al., 2004). The positive-strand RNA progeny is transcribed in a five to ten fold in excess compared to negative-strand RNA. NS5B RpRd was initially thought to catalyze primer-dependent initiation of RNA synthesis, either through elongation of a primer hybridized to the RNA template or through a copy-back mechanism (Behrens et al., 1996). More recently, the HCV RdRp was shown to be capable of initiating de novo RNA synthesis under certain experimental conditions (Zhong et al., 2000).

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Initiation of RNA strand synthesis at the 3'-end of the plus and minus strands involves domain I of the 5'UTR, which can form a G/C-rich stem-loop, the 3'UTR and a cis-acting replication element (5BSL3.2) consisting of 50 bases located in a large predicted cruciform structure at the 3' end of the HCV NS5B-coding region (You et al., 2004). Initiation of RNA replication is triggered by an interaction between proteins of the replication complex, the 3'X region of the 3'UTR, and 5BSL3.2 that forms a pseudoknot structure with a stem-loop in the 3'UTR (Astier-Gin et al., 2005; Friebe et al., 2005; You et al., 2004). A phosphorylated form of PTB was found in the replication complex and PTB was shown to interact with two conserved stem-loop structures of the 3'UTR, an interaction thought to modulate RNA replication (Chang and Luo, 2005; Luo, 1999; Luo, 2004). Importantly, inhibition of PTB expression by means of small interfering RNAs reduced the amount of HCV proteins and RNA in HCV replicon-harboring Huh7 cells (Chang and Luo, 2005).

VIRUS ASSEMBLY AND RELEASELittle is known about HCV assembly and release due to the lack of appropriate study models. Different variants of the HCV core protein, which can exist as dimeric, and probably multimeric forms as well, have been shown to be capable of self assembly in yeast in the absence of viral RNA, generating virus-like particles with an average diameter of 35 nm (Acosta-Rivero et al., 2004a; Acosta-Rivero et al., 2004b). Recent reports suggested that the N-terminal portion of the core protein is sufficient for capsid assembly, in particular the two clusters of basic residues (Klein et al., 2005; Klein et al., 2004; Kunkel et al., 2001; Lorenzo et al., 2001; Majeau et al., 2004). In bacterial systems, HCV core proteins efficiently self-assembled to yield nucleocapsid-like particles with a spherical morphology and a diameter of 60 nm, but the presence of a nucleic acid was required (Kunkel et al., 2001). Overall, particle formation is probably initiated by the interaction of the core protein with genomic RNA; HCV core can indeed bind positive-strand RNA in vitro through stem-loop domains I and III and nt 23-41 (Shimoike et al., 1999; Tanaka et al., 2000). It is tempting to speculate that the core-RNA interaction may play a role in the switch from replication to packaging.

Virus-like particles were produced in mammalian cells by using a chimeric virus replicon allowing high-level expression of HCV structural proteins in BHK-21 cell lines (Blanchard et al., 2002). Budding of virus-like particles of 50 nm in diameter in the dilated ER lumen was observed (Blanchard et al., 2003). Transfection of full-length HCV RNA in HeLa G and HepG2 cell lines led to the formation of virus-like particles with a diameter of 45 to 60 nm, which were synthesized and assembled in the cytoplasm and budded into the ER cisternae to form coated particles (Dash et al., 1997; Mizuno et al., 1995). Indeed, the HCV envelope glycoproteins E1 and E2 associate with ER membranes through their transmembrane domains


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(Cocquerel et al., 1998), suggesting that virus assembly occurs in the ER. Structural proteins have been detected both in the ER and the Golgi apparatus, suggesting that both compartments are involved in later maturation steps (Serafino et al., 2003). Moreover, the presence of N-glycan residues at the surface of HCV particles is also in keeping with a transit via the Golgi apparatus. The mechanisms underlying exportation of mature virions in the pericellular space have yet to be understood. Newly produced virus particles may leave the host cell by the constitutive secretory pathway.

STRUCTURE OF HCV VIRIONSHCV is thought to adopt a classical icosahedral scaffold in which glycoproteins E1 and E2 are anchored to the host cell-derived double-layer lipid envelope. Within the envelope is the nucleocapsid which is likely composed of multiple copies of the core protein, forming an internal icosahedral viral coat that encapsidates the viral genomic positive-strand RNA. EM and immuno-EM (IEM) studies of bona fide HCV particles have been hampered by the low amount of viruses in blood and tissues, the failure to efficiently propagate HCV in cell culture, the poor sensitivity of these methods, and antibody cross-reactivity. Visualization of HCV virions or virus-like particles was therefore made essentially from in vitro or non-human in vivo models.

Infection of primary cells or stable cell lines of hepatic or lymphoid origin with sera from HCV-infected patients revealed the presence of spherical virus-like particles (Lacovacci et al., 1997; Serafino et al., 1997; Shimizu et al., 1996). Transfection of Huh7 cells with full-length HCV genomes did not lead to virion production (Pietschmann et al., 2002), but virus-like particles were generated after transfection of HepG2 or Hela G cells (Dash et al., 1997; Mizuno et al., 1995). HCV virus-like particles could also be produced in mammalian cells, by means of recombinant Semliki Forest virus (SFV) or vesicular stomatitis virus (VSV) replicons expressing genes encoding the structural HCV proteins (Blanchard et al., 2003; Ezelle et al., 2002), and in insect cells infected with a recombinant baculovirus expressing HCV structural proteins (Baumert et al., 1998; Luckow and Summers, 1988; Maillard et al., 2001).

MORPHOLOGY OF HCV PARTICLESEarly filtration studies performed in sera from chimpanzees with non-A, non-B hepatitis suggested that the diameter of the causal agent was in the order of 30-60 nm (He et al., 1987; Yuasa et al., 1991). EM and IEM analysis of particles recovered from the blood and liver of infected chimpanzees and patients revealed the presence of spherical particles of 33-70 nm (Bosman et al., 1998; Ishida et al., 2001; Jacob et al., 1990; Kaito et al., 1994; Li et al., 1995; Petit et al., 2003). Detergent treatment of infectious sera yielded 30-40 nm icosahedron-shaped particles containing both

25


the HCV core protein and HCV RNA (Takahashi et al., 1992). Virus-like particles of 45-60 nm was observed in the supernatant of primary cells or stable cell cultures treated with infectious sera and of cell lines transfected with the full-length HCV ORF (Dash et al., 1997; Iacovacci et al., 1997; Mizuno et al., 1995; Serafino et al., 1997; Shimizu et al., 1996). HCV-like particles of 20-60 nm in diameter were also produced by the expression of HCV structural proteins in cell-free systems (Klein et al., 2004), SFV replicons (Blanchard et al., 2002; Blanchard et al., 2003), VSV vectors in rodent BHK-21 cells (Ezelle et al., 2002), bacterial systems (Kunkel et al., 2001; Lorenzo et al., 2001), baculovirus vectors in insect cells (Baumert et al., 1998; Xiang et al., 2002) and yeast expression vectors (Acosta-Rivero et al., 2001; Acosta-Rivero et al., 2004b; Falcon et al., 1999).

The recently developed cell culture system is capable of producing large amounts of infectious HCV virions (Lindenbach et al., 2005b; Wakita et al., 2005; Zhong et al., 2005). Two types of viral particles could be visualized in IEM: particles of 30-35 nm in diameter likely to correspond to the viral nucleocapsids, and particles of 50-60 nm in diameter likely to be the infectious virions (Fig. 4) (Wakita et al., 2005).

Fig. 4. HCV viral particle produced in a tissue culture system from a cloned viral genome (Wakita et al., 2005). Viral particles were generated after transfection of the human hepatoma cell line Huh7 by HCV replicons of the JFH1 genotype 2a strain cloned from a Japanese patient with fulminant hepatitis (see Chapter 16). HCV particles had a density of 1.15-1.17 g/ml and a spherical morphology with an average diameter of approximately 55 nm. They were infectious for chimpanzees (Wakita et al., 2005). The photograph is a courtesy of Ralf Bartenschlager.


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CIRCULATING FORMS OF HCV VIRIONS

PLASMA COMPARTMENTALIZATION OF HCV PARTICLES

HCV was initially reported to have a lower buoyant density than other members of the Flaviviridae family on 20-60% isopycnic sucrose density gradients (1.05 to 1.07 g/ml vs 1.15 to 1.25 g/ml, respectively) (Lindenbach and Rice, 2001; Trestard et al., 1998; Yoshikura et al., 1996). Ultracentrifugation of sera from patients with acute and chronic HCV infection revealed the presence of two populations of HCV particles with a broad range of densities, from 1.06 to 1.25 g/ml. Low-density HCV particles were shown to be principally associated with lipids and lipoproteins and to contain the infectious virus, whereas high-density HCV particles were largely associated with immunoglobulins in the form of immune complexes and supposedly less infectious (Aiyama et al., 1996; Andre et al., 2002; Dienstag et al., 1979; Hijikata et al., 1993; Thomssen et al., 1992). Interestingly, the respective proportions of high- and low-density fractions in infected patients' blood were reported to fluctuate over the course of infection and according to the stage of liver disease (Choo et al., 1995; Hijikata et al., 1993; Kanto et al., 1994; Kanto et al., 1995; Petit et al., 2003).

NON-ENVELOPED NUCLEOCAPSIDS

The existence of non-enveloped HCV nucleocapsids during natural infection and their role in the pathophysiology of HCV infection has been debated. Lipo-viro-particles (LVPs) rich in HCV RNA, HCV core protein, triglycerides and apoproteins (especially apoB and apoE) were recently described as large spherical particles of 100 nm, the delipidation of which yielded capsid-like structures (Andre et al., 2002). Non-enveloped nucleocapsids were detected in the serum of infected patients and in hepatocytes from patients and experimentally infected chimpanzees (Falcon et al., 2003a; Falcon et al., 2003b; Maillard et al., 2001). Non-enveloped HCV particles recovered from the plasma of infected individuals had a buoyant density of 1.27 to 1.34 g/ml (Maillard et al., 2001). They were heterogeneous in size, with a diameter of 38-62 nm in EM, and were recently shown to exhibit Fcγ receptor-like activity and bind non-immune IgG (Maillard et al., 2001; Maillard et al., 2004). Whether or not non-enveloped nucleocapsids are infectious remains to be established.

CONCLUSIONThe development of novel anti-HCV therapeutic agents has been stymied by the lack of an efficient in vitro viral infection system and a suitable animal model. Although significant progress has been made through genetic and biochemical approaches in dissecting the molecular processes of HCV replication, our understanding of the viral entry and virion production steps remains rudimentary. Furthermore, HCV exists as "quasispecies" in patients due to its high mutation rate and thus viral resistance will likely be a problem for the emerging small-molecule HCV inhibitors (Pawlotsky, 2003; Pawlotsky, 2006). The recent development of a robust cell culture

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system for HCV infection may unravel new aspects of HCV replication, which in turn will facilitate the development of specific antivirals that target each stage in the virus life cycle.

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Chapter 2

HCV 5' and 3'UTR: When Translation Meets Replication

Stephanie T. Shi and Michael M. C. Lai

ABSTRACTSimilar to other positive-strand RNA viruses, the non-coding regions of HCV RNA, referred herein as 5' and 3' untranslated regions (5'UTR and 3'UTR), contain important sequence and structural elements critical for HCV translation and RNA replication. The 5'UTR harbors an internal ribosome entry site (IRES) that directs viral protein translation via a cap-independent mechanism. As the initiation sites for RNA synthesis, both 5'UTR and 3'UTR contain signals that are indispensable for and regulate viral RNA replication. Additional structural elements involved in translation or RNA replication are also present in both ends of the protein (core and NS5B)-coding regions. These RNA elements interact with each other either directly or through the binding of viral and cellular proteins that are most likely involved in the regulation of translation and RNA replication processes. Since RNA replication and translation occur on the same RNA molecule, mechanisms must exist to regulate and separate these two processes. This chapter details the current understanding of the roles of the UTRs and other structural components in the viral RNA as well as their binding proteins in HCV translation and RNA replication and speculate on the possible mechanisms regulating these two different processes.

INTRODUCTIONHCV is a typical flavivirus containing a single-stranded, positive-sense RNA of 9.7 kb in length (Choo et al., 1991). The viral RNA contains a single large open reading frame (ORF) flanked by an untranslated region (UTR) at each end, a genomic organization conserved among members of the Flaviviridae family. One of the most important features of HCV RNA is its high degree of genetic variability as a result of mutations that occur during viral replication. However, the mutation rate varies significantly in the different regions of the HCV genome, of which the 5'UTR and the extreme end of the 3'UTR have the lowest sequence diversity among various genotypes and subtypes (Choo et al., 1991; Miller and Purcell, 1990; Muerhoff et al., 1995). The relatively conserved nature of these regions signifies their functional importance in the viral life cycle.

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A combination of phylogenetic analysis, computer modeling, and chemical and enzymatic probing has enabled the identification of structural elements in the 5' and 3' UTRs of HCV RNA. The viral RNA elements (internal ribosome entry site, IRES) critically involved in the cap-independent translation of HCV RNA have been analyzed extensively. In contrast, the study of the mechanism of HCV RNA replication was more limited due to the lack of efficient cell culture or small animal models. The generation of consensus cDNA clones that are infectious in chimpanzees provided the first tools for molecular genetic analysis of HCV RNA replication (Beard et al., 1999; Choo et al., 1989; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1999a; Yanagi et al., 1998). Using this approach, the regions in the 3'UTR that are required for viral replication have been identified (Kolykhalov et al., 1997; Yanagi et al., 1999b). More recently, the development of and advances in the cell-based subgenomic replicon system have identified additional RNA elements of the UTRs and other cis-acting replication elements (CREs) that are involved in RNA replication and translation (Friebe et al., 2005; Friebe et al., 2001; Lee et al., 2004a; You et al., 2004).

A number of viral and cellular proteins have been shown to interact with the essential structural elements in the non-coding and coding regions of HCV RNA and are presumably involved in the regulation of the viral translation and/or RNA replication processes. The precise functional roles of most of these proteins have not been established. The recent development of cell-free HCV RNA replication systems (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) provides an additional tool for studying the viral and host proteins involved in the translation and replication of HCV RNA, thus identifying novel targets for the development of more effective antiviral therapies.

STRUCTURAL AND FUNCTIONAL COMPONENTS OF THE HCV RNAThe 5'UTR and the extreme end of the 3'UTR are the most conserved regions of HCV RNA in terms of primary sequence and secondary structures. Together with the fact that these structured domains are located at the 5' and 3' ends of the genome, it stands to reason that they play important roles in viral RNA translation and/or replication.

5'UTRThe 5'UTR of the HCV genome is 341-nt long in most viral isolates. There is more than 90% sequence identity among different HCV genotypes, with some segments nearly identical among different strains (Bukh et al., 1992). The secondary and tertiary structures of this region are also largely conserved (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a). The 5'UTRs of HCV, GBV-B (Muerhoff et al., 1995), and pestiviruses, such as bovine viral diarrhea virus (BVDV) and

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classical swine fever virus, share extensive homology in primary sequence and secondary structure (Brown et al., 1992; Han et al., 1991; Honda et al., 1996a; Simons et al., 1995), signifying GBV-B and pestiviruses as the closest relatives to HCV (Ohba et al., 1996). A combination of computational, phylogenetic, and mutational analyses of the HCV 5'UTR has identified four major structural domains (domains I-IV) (Fig. 1), most of which are also conserved among HCV genotypes, GBV-B, and pestiviruses (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a; Smith et al., 1995). Common features include a large stem-loop III and a pseudoknot (psk). The 5'UTR sequences of HCV and GBV-B have two smaller stem-loops, stem-loop Ia near the extreme 5' end and stem-loop IV containing the translation initiation codon (Honda et al., 1996a).

Fig. 1. The structures of the 5'UTR (Rijnbrand and Lemon, 2000) and 3'UTR (Ito and Lai, 1997; Kolykhalov et al., 1996) of HCV RNA (represented by the HCV-H strain). The structural diagram of the 5'UTR was kindly provided by Drs. René Rijnbrand and Stanley Lemon. psk, pseudoknot. The start codon (nt 342) and stop codon are indicated by boldface characters. The shaded boxes in 5'- and 3'-UTR and are RNA elements putatively involved in RNA replication. The enclosed RNA sequences in 5'-UTR are the reported elements required for efficient IRES-dependent translation.

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The first 40 nt of the 5'UTR constitutes domain I, which is involved in RNA replication but not essential for translation; therefore, the function of this region is distinct from the rest of the 5'UTR, which is critical for translation (Friebe et al., 2001; Luo et al., 2003). The remaining domains II-IV constitute an IRES (Fig. 1) (Brown et al., 1992), which mediates the cap-independent translation of the HCV ORF (Tsukiyama-Kohara et al., 1992). Domains II and III are relatively more complex than domain IV and contain multiple stems and loops (Honda et al., 1999a; Lemon and Honda, 1997). Several electron microscopy (Beales et al., 2001; Spahn et al., 2001) and NMR studies (Lukavsky et al., 2000) have provided detailed structural information on the main domains of the IRES. Domains IIIa–IIIc and II extend in opposite directions from a small central domain that includes stem loops and junctions IIIe–IIIf (Spahn et al., 2001). The hairpin loop of the small IIIe subdomain forms a novel tetraloop fold with three exposed Watson–Crick faces that may be involved in 40S ribosome binding (Lukavsky et al., 2000). The stem of subdomain IIId forms a loop E motif similar to those observed in prokaryotic and eukaryotic ribosomal RNA, and a six-nucleotide hairpin loop containing an S-turn motif (Klinck et al., 2000; Lukavsky et al., 2000). The sequences of the hairpin loops of subdomains IIIe and IIId are conserved among all HCV isolates and play an important role in translation initiation.

The base of domain III forms a highly conserved pseudoknot, which is critical for IRES activity (Wang et al., 1995). Similar pseudoknots with almost identical primary sequences also exist in the pestiviral and GBV-B IRES elements (Lemon and Honda, 1997). The pseudoknot is part of the binding site for the 40S ribosome subunit (Kolupaeva et al., 2000). Another tertiary structural element in domain II, identified by RNA-RNA crosslinking, may also be involved in ribosome binding (Lyons et al., 2001). Domain IV is composed of a small stem-loop (stem-loop IV) in which the initiator codon AUG is located within the single-stranded loop region (Honda et al., 1996a). Stem-loop IV is not required for internal entry of ribosomes. In fact, the stability of this stem-loop structure is negatively correlated with the translation efficiency of the viral RNA (Honda et al., 1996a).

According to a structure-based classification scheme originally designed for picornaviral IRES elements (Wimmer et al., 1993), the HCV IRES, together with the IRES elements of the closely related pestiviruses and GBV-B, is classified into type 3 of four existing types (Lemon and Honda, 1997). The picornaviral and flaviviral IRES elements are significantly different in a number of aspects, suggesting distinct mechanisms of translation initiation for these two virus families (Rijnbrand and Lemon, 2000). The picornaviral IRES elements have been shown to be more efficient than the HCV IRES in directing translation (Borman et al., 1995). In contrast, viruses in the genus Flavivirus (e.g. yellow fever virus) have significantly shorter 5' UTRs with a cap structure, m7GpppN1mpN2 (Westaway, 1987).

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3'UTRThe 3'UTR of HCV varies between 200 and 235 nt long, which typically consists of three distinct regions, in the 5' to 3' direction, a variable region, a poly(U/UC) stretch, and a highly conserved 98-nt X region (Blight and Rice, 1997; Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996; Yamada et al., 1996). The variable region follows immediately the termination codon of the HCV polyprotein, and is variable in length (ranging from 27 to 70 nt) and composition among different genotypes. However, it is highly conserved among viral strains of the same genotype (Kolykhalov et al., 1996; Yanagi et al., 1997; Yanagi et al., 1998). Computer analysis has identified two possible stem-loop structures in the variable region, with the first stem-loop extending into the 3' end of the NS5B-coding sequence (Han and Houghton, 1992; Kolykhalov et al., 1996). The poly(U/UC) tract consists of a poly(U) stretch and a C(U)n-repeat region (referred to as the transitional region) and varies greatly in length and slightly in sequence among different viral isolates (Tanaka et al., 1996). The transitional regions of genotypes 2a, 3a, and 3b have several conserved A residues, which are not present in genotypes 1b and 2b (Tanaka et al., 1996; Yamada et al., 1996; Yanagi et al., 1999a). The presence of the polypyrimidine tract within the 3'UTR is unique to HCV and GBV-B (Simons et al., 1995) among flaviviruses. The length of this region has been correlated with the replication capability of HCV RNA (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). The X region forms three stable stem-loop structures that are highly conserved across all genotypes (Blight and Rice, 1997; Ito and Lai, 1997; Kolykhalov et al., 1996) (Fig. 1). A recent study of the structure of the X region by chemical and enzyme probing has confirmed the presence of SL1 and SL3, but proposed that the region between the two stem-loops folds into two hairpins instead of one and may further form a hypothetical pseudoknot (Dutkiewicz and Ciesiolka, 2005). On the other hand, the complementary sequence of the X region in this region forms a 3-stem-loop structure (Dutkiewicz and Ciesiolka, 2005). There is no poly(A) sequence in the 3'UTR. Instead, the 3'UTR sequence, particularly the X region, is involved in the regulation of translation, much in the same way as the poly(A) sequence in the mRNAs of other RNA viruses. Conceivably, these sequences are involved in the replication, stabilization and also packaging of viral RNA.

As a result of the stem-loop formation in the X region, the HCV genome is predicted to end with a double-stranded stem. Examination of the 3'-terminal sequences of the HCV genome in sera from infected patients revealed that most HCV RNAs contain identical 3' ends with no extra sequence downstream of the X tail (Tanaka et al., 1996). However, one particular cDNA clone derived from a patient's serum did contain 2 additional nt (UU), thus generating a single-stranded tail (Yamada et al., 1996). The structure of the exact 3'-end will have implications for the initiation of RNA replication.

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OTHER STRUCTURAL COMPONENTS IN THE PROTEIN-CODING REGIONBioinformatic analysis has revealed the possible presence of additional secondary structures in other parts of the HCV genome (Hofacker et al., 1998). These include possible secondary structures in the core- and NS5B-coding regions (Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002). Consistent with the importance of the predicted secondary structures, it has been shown that synonymous nucleotide mutations are suppressed in the core- and NS5B-coding regions and that compensatory mutations are frequently observed within the predicted stems (Ina et al., 1994; Smith and Simmonds, 1997).

The predicted secondary structures within the core-coding region encompass the first 14 nts of the core gene, which form part of the IRES stem-loop IV (Lemon and Honda, 1997). There are two more stem-loops between nt 47 and 167 of the core-coding sequence (nt 391-511 of the genome), which is conserved among all six HCV genotypes (Smith and Simmonds, 1997). This region, corresponding roughly to nt 408-929, has been shown to interact with the 5'UTR, resulting in the reduction of HCV IRES-mediated translation (Honda et al., 1999b). In the NS5B-coding region (Hofacker et al., 1998; Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002; You et al., 2004), six potential stem-loop structures have been predicted based on computer modeling (You et al., 2004). The functional significance of five of these structures in RNA replication has been implicated from mutational analysis and RNA structure probing in the context of the subgenomic replicon. Of particular interest is a cruciform structure (5BSL3) at the 3' terminus of NS5B, which contains three major stem-loop structures, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Its involvement in RNA replication will be discussed in a later section.

HCV TRANSLATIONTranslation of the polyprotein from the HCV RNA genome is the first macromolecular synthetic event after the viral RNA is released into the cytoplasm of host cells. It is carried out by a cap-independent mechanism mediated by the highly structured HCV IRES. The HCV genomic RNA serves as an mRNA for the translation of a single polyprotein, which is processed by cellular and viral proteases into at least 10 structural and nonstructural proteins (De Francesco et al., 2000).

STRUCTURAL COMPONENTS REQUIRED FOR TRANSLATIONThe first 40 nts of the HCV RNA genome, including the first stem-loop domain (domain I), are not required for translation (Honda et al., 1996b; Rijnbrand et al., 1995). Instead, deletion of this domain resulted in a stimulation of translation of a heterologous reporter RNA (Yoo et al., 1992). However, in the context of the HCV subgenomic replicon, deletion of this domain reduced protein expression by 3 to 5 fold (Luo et al., 2003). In addition, a dinucleotide sequence at nt 34-35 has been shown to contribute to the differential translation efficiencies between genotype

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1a and 1b isolates (Honda et al., 1999b). It is, therefore, possible that domain I is also involved in the regulation of HCV translation in some fashions. The primary element of the IRES starts at nt 44, which coincides with the 5' border of domain II (Honda et al., 1999a; Honda et al., 1996b; Reynolds et al., 1995; Rijnbrand et al., 1995). However, the precise 3' border of the IRES is controversial.

The stem-loop IV of the 5'UTR is predicted to extend into the coding region to include the first 10 nts (nt 345-354) of the core-encoding gene. Indeed, several studies have reported the requirement for a short sequence (up to 30 nt) in the core-coding region for optimal IRES function (Honda et al., 1996a; Hwang et al., 1998; Lu and Wimmer, 1996; Reynolds et al., 1996). However, efficient translation has also been observed with certain reporter genes fused immediately after the start codon, without the core protein-coding sequences (Tsukiyama-Kohara et al., 1992; Wang et al., 1993). The differences in the conclusions may have been due to the assay systems and heterologous reporters employed. It has been found that expression of the reporter gene secretory alkaline phosphatase, but not that of chloramphenicol acetyltransferase, depends on the presence of downstream core-coding sequences (Rijnbrand et al., 2001). Conceivably, the core-coding region may contribute to IRES function by preventing undesirable base pairing of the IRES with other inhibitory sequences or by promoting favorable protein binding to the IRES. This core-coding

Fig. 2. Cis-acting RNA replication regulatory elements in the NS5B-coding region that interact with the 3'UTR (represented by the HCV-Con1 strain). (A) The cruciform structure formed at the end of the NS5B-coding sequence contains 5BSL3.1, 5BSL3.2, and 5BSL3.3, among which 5BSL3.2 is required for HCV RNA replication (You et al., 2004). (B) Kissing-loop interaction between the loop sequences of 5BSL3.2 and SL2 of the X region (Friebe et al., 2005).

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region contains an adenosine-rich stretch, which has been shown to recruit a cellular protein that enhances the HCV IRES activity (Kim et al., 2004a; Reynolds et al., 1995). So far, nt 354 is generally regarded as the consensus 3' boundary of the IRES (Honda et al., 1999a), but the sequence immediately downstream of the IRES (up to nt 371) may have a stimulating effect on IRES-directed translation. Interestingly, the core-coding sequences further downstream (near the C-terminal portion) have been shown to play a negative-regulatory role in HCV translation (Ito and Lai, 1999; Kim et al., 2003; Wang et al., 2000).

Besides the 5'UTR, the 3'UTR sequences, particularly the X region, may also play a role in HCV RNA translation. It has been shown that HCV RNA containing the X region was translated 3- to 5-fold more efficiently than the corresponding RNAs without this region (Ito et al., 1998). The enhancement of IRES-dependent translation by 3'UTR may be mediated by polypyrimidine tract-binding protein (PTB), which binds to both the 5' and 3'UTR (Ali and Siddiqui, 1995; Ito and Lai, 1997; Tsuchihara et al., 1997). Since PTB can interact with itself, it can potentially mediate circularization of HCV RNA, thereby enhancing translation. The role of the 3'UTR in translation is reminiscent of the poly(A) tail and the poly(A)-binding protein in the translation of poly(A)-containing mRNAs (Kahvejian et al., 2001). However, a different study reported that deletion of the poly(U/UC) tract or the stem-loop 3 of the X region resulted in an enhancement of translation efficiency; the increase in translation was not mediated by PTB (Murakami et al., 2001). Additional studies are required to understand the role of the 3'UTR in IRES-mediated translation of HCV proteins.

THE HCV TRANSLATION MACHINERYThe HCV IRES is responsible for directing the 40S ribosomal subunit in close contact with the start codon for translation initiation (Lemon and Honda, 1997; Wang et al., 1993). Enzymatic and chemical footprinting and domain-deletion experiments have identified domain II and the basal part of domain III, excluding domain IIIb, as the binding site for the 40S ribosome subunits (Kieft et al., 2001; Kolupaeva et al., 2000; Lukavsky et al., 2000; Pestova et al., 1998). Although the HCV IRES with or without domain II recruits the 40S ribosome subunit with comparable efficiency (Otto et al., 2002), interaction of domain II with the 40S subunit induces or stabilizes the conformational changes within the ribosome and facilitates the 3´ end of the coding RNA to thread into the mRNA entry channel (Spahn et al., 2001). The GGG triplet (nt 266-268) of the hexanucleotide (UUGGGU) apical loop of stem-loop IIId and the pseudoknot are essential for ribosome binding (Kolupaeva et al., 2000). Mutagenesis studies have also confirmed that the GGG triplet is essential for IRES activity both in vitro and in vivo (Jubin et al., 2000).

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The viral 5'UTR forms a binary complex with the 40S ribosomal subunit in the absence of any canonical or non-canonical initiation factors (Pestova et al., 1998). A ribosomal protein S5, in particular, is important for the efficient translation initiation of HCV RNA (Fukushi et al., 1997; Fukushi et al., 2001b; Pestova et al., 1998). Blocking of the S5 binding to HCV IRES interfered with efficient ribosome assembly at the translation initiation site (Ray and Das, 2004). These features suggest that HCV IRES uses the prokaryotic mode for forming the mRNA-40S ribosome complex (Pestova et al., 1998).

Several basal translation initiation factors have been reported to be involved in the HCV IRES-mediated translation. The eukaryotic initiation factor-3 (eIF3), alone or together with the 40S ribosome subunit and the eIF2-GTP-initiator tRNA complex, can specifically interact with the HCV IRES stem-loop IIIb in the absence of eIF4A, eIF4B and eIF4F, which are required for ribosomal binding during cap- or EMCV IRES-dependent translation (Kieft et al., 2001; Kolupaeva et al., 2000; Pestova et al., 1998; Sizova et al., 1998). eIF3 binding is not necessary for 40S-HCV IRES assembly, but is essential for the joining of 60S subunit to form the active 80S ribosomal complexes (Pestova et al., 1998). These findings suggest that HCV employs a modified mechanism of IRES-dependent translation. Rabbit reticulocyte lysates depleted of certain translation factors, such as eIF4G, cannot support foot-and-mouth-disease virus IRES-, but still can support HCV IRES-dependent translation (Stassinopoulos and Belsham, 2001). eIF2Bγ and eIF2γ have also been identified as cofactors of HCV IRES-mediated translation by a functional genomics approach (Krüger et al., 2000), although their roles in translation have not been established. These findings combined indicate that HCV IRES-dependent translation employs a prokaryotic mode for assembling RNA-ribosome complex and requires only a minimum set of canonical translation factors.

HCV RNA REPLICATIONBy analogy with other members of the Flaviviridae, HCV is presumed to replicate its genome through the production of a full-length negative-strand RNA. Positive-strand RNAs are then synthesized from the negative-strand template in five- to ten-fold molar excess over the negative-strand RNA (Lohmann et al., 1999) to be used in translation, replication, and packaging into progeny viruses. Since RNA replication has to initiate from the 3'- end of the RNA template of both strands, the corresponding 5' and 3' UTR of HCV RNA genome likely contains the sequences required for the initiation and/or regulation of RNA replication.

STRUCTURAL COMPONENTS REQUIRED FOR RNA REPLICATIONSince the 5'UTR is involved in the initiation of both translation and RNA replication, any possible effects of this region on translation will impact RNA replication indirectly and vice versa. Therefore, the direct role of 5'UTR in RNA replication

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is difficult to assess. Separation of RNA replication and translation was initially achieved by inserting the IRES elements of poliovirus or classical swine fever virus between the serially deleted 5'UTR of HCV and the ORF (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). The deletions introduced into the 5'-terminal 40 nt upstream of the IRES region abolished RNA replication but only moderately affected translation. The first 125 nt of the HCV genome, which includes domain I and II of the 5'UTR, was shown to be essential and sufficient for RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). This region overlaps with the 5'end of the IRES. The replication efficiency of RNA was tremendously increased by the inclusion of the complete 5'UTR (Friebe et al., 2001). Compared with its close relative BVDV, the requirements for RNA sequences or structures within the 5'UTR of HCV appear to be more complex because much longer sequences or particular structures within the IRES are necessary for efficient RNA replication (Frolov et al., 1998; Wilhelm Grassmann et al., 2005). However, further studies are required to show whether the sequences downstream are directly involved in RNA replication or merely contribute to the preservation of the structural and functional integrity of the minimal replication signal.

Consistent with a role for the 3' terminal nt of the viral RNA in the initiation of negative-strand RNA, the 3'UTR sequences have been shown to play an essential role in HCV RNA replication in vitro (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a) and in vivo (Kolykhalov et al., 2000; Yanagi et al., 1999b). The 3'UTR sequences were first shown to be required for the replication of HCV RNA when deletion of the 3' terminal sequences destroyed the ability of otherwise infectious synthetic genome-length HCV RNA to initiate infection in intrahepatically inoculated chimpanzees (Kolykhalov et al., 2000; Yanagi et al., 1999b). Using a subgenomic HCV replicon, the 3' terminal RNA signals required for HCV RNA replication were determined to be approximately 225 nt from the 3' end of the genome (Yi and Lemon, 2003a). The 3'-most 150 nt of the genome, which includes the 98-nt X region and the 52 nt of the poly(U/UC) tract, are essential for replication of HCV RNA, while the remaining upstream region of the 3'UTR plays a facilitating role (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b). These results suggest an interesting symmetry in the 5'- and 3'- terminal RNA replication signals since the 5'-most domains I and II of the 5'UTR are essential for replication, while sequences lying further downstream within the 5'UTR help to facilitate replication but are not absolutely required (Friebe et al., 2001; Kim et al., 2002b). The X region interacts with the recombinant HCV RNA polymerase (Cheng et al., 1999; Oh et al., 2000), although other parts of the 3' end of HCV genome may contain additional NS5B-binding sites (Cheng et al., 1999). The NS5B-binding domain within the X region has been mapped to stem II and the single-stranded region connecting stem-loops I and II (Oh et al., 2000). Truncation of 40 nts or more from the 3' end of the X region abolished its template

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activity in vitro (Oh et al., 1999; Oh et al., 2000). A more extensive mutational analysis of the 3'-end 46 nt that form the terminal hairpin (stem-loop I) in the HCV replicon provided strong functional evidence for the existence of the structure and for an essential role of the structure in the replication of HCV RNA (Yi and Lemon, 2003b). It is interesting that the X region is also necessary for efficient translation of HCV protein (Ito et al., 1998); thus, the same set of sequences are involved in both RNA replication and translation.

The poly(U/UC) tract is required for HCV RNA replication (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). It is possible that this region assists in circularizing the viral genome, which has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). This sequence binds several cellular proteins (e.g. PTB), which may mediate RNA-RNA interaction (Ito and Lai, 1999) and/or the binding of the replicase complex to RNA. The length of the poly(U/UC) region may influence viral replication as HCV RNA with a longer poly(U/UC) region had a replicative advantage in chimpanzees (Kolykhalov et al., 1997; Yanagi et al., 1999b) than the one with a shorter poly(U/UC). Similar observation was made in the subgenomic replicon RNAs (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a). Conversely, the poly(U/UC)-rich sequence may serve as a modulator of RNA replication under some conditions, as shown in an in vitro RNA polymerase reaction, in which HCV RNA polymerase stutters at this region (Oh et al., 1999).

The sequences within the variable region of the 3'UTR are not essential for RNA replication (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b; Yi and Lemon, 2003a), a finding similar to those of other flaviviruses (Khromykh and Westaway, 1997; Mandl et al., 1998; Men et al., 1996). Interruption of sequence integrity within this region by insertion of the extraneous sequences in this region did not interfere with the replication of the HCV RNA or replicons (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b). Nevertheless, deletions in this region impaired the efficiency of amplification of subgenomic replicons (Yi and Lemon, 2003a).

Some of the conserved RNA elements identified in the NS5B-coding region may serve as recognition sites for the HCV replicase complex since partially purified NS5B specifically binds to the coding sequences of NS5B RNA (Cheng et al., 1999), but their involvement in RNA replication has not been established until recently. The NS5B-coding region contains a predicted cruciform structure (5BSL3) consisting of three stem-loops, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Mutations disrupting the 5BSL3.2 blocked RNA replication, whereas 5BSL3.1 and 5BSL3.3 were shown not to be required for RNA replication (Friebe et al., 2005; You et al., 2004). Insertion of 5BSL3.2 alone into the variable region of the 3'UTR was sufficient to rescue RNA replication of a replicon in which all three 5BSLs in the

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NS5B-coding region were disrupted, indicating that 5BSL3.2 can act as a cis-acting RNA replication element. This insertion allowed the analysis of individual elements within 5BSL3.2 in more detail without the complication of introducing amino acid changes in the NS5B-coding region (Friebe et al., 2005).

5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop; the stem structures, but not their primary sequences, are required for RNA replication (You et al., 2004). In addition, a kissing-loop interaction between a 7-nt-long complementary sequence in 5BSL3.2 and SL2 in the X region has been proven essential for RNA replication (Friebe et al., 2005). In the upper loop of 5BSL3.2, a CACAGC sequence motif is found to be virtually invariant among HCV genotypes and is also present in cis-acting RNA sequences of distantly related flaviviruses, such as Kunjin virus, West Nile virus, or Dengue virus (Markoff, 2003). Given the high genetic conservation in this particular region of the genome, it may be speculated that certain ubiquitously expressed and evolutionarily conserved host cell proteins are involved in the formation of a replication complex that interacts with the 3' end of the flavivirus genome.

INITIATION OF NEGATIVE- AND POSITIVE-STRAND RNA SYNTHESISRecombinant NS5B proteins are capable of primer-independent initiation of RNA synthesis on a variety of virus-specific and nonspecific RNA templates in vitro (Ferrari et al., 1999; Lohmann et al., 1998; Oh et al., 1999). However, there are conflicting descriptions of the precise initiation site of negative-strand RNA transcription on the HCV-specific templates (Hong et al., 2001; Kim et al., 2002a; Oh et al., 2000; Shim et al., 2002). Oh et al. reported that the transcription of the negative-strand RNA was initiated within the loop sequence of the 3'X stem-loop I, at approximately 21 nt from the 3' end of the RNA (Oh et al., 2000). Kim et al. reported that transcription initiated further downstream, within the 3' stem sequence of SL1 (Kim et al., 2002a). Shim et al. has shown that transcription can be initiated by a recombinant NS5B polymerase in vitro at the 3' end of short oligonucleotide templates representing the 3' terminus of the positive-strand genomic RNA (Shim et al., 2002). The terminal U is preferred as the initiation nt (Shim et al., 2002), which is confirmed by the study of a subgenomic replicon (Yi and Lemon, 2003b). Hong et al. proposed that a unique β-hairpin within the thumb domain of the NS5B polymerase positions the terminal sequences of the genome so as to initiate de novo transcription from the 3' terminal nucleotides (Hong et al., 2001). It was proposed that the β-hairpin ensures the initiation of de novo RNA synthesis at the 3' terminus by preventing movement of the 3' end of the single-stranded RNA template into the active site of the enzyme. Conceivably, the presence of other viral and cellular proteins may affect the selection of the initiation point of RNA replication in vivo.

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The initiation of the positive-strand RNA synthesis has not been as well studied. Conceivably, the 3' terminus of the negative-strand RNA is essential for positive-strand RNA replication. In vitro replication studies using recombinant NS5B showed that the minimal RNA fragment required for efficient replication of the negative-strand RNA spans nt –239 to –1 (Oh et al., 1999), which is complementary to domains I to III of the 5'UTR. Various site-specific mutation studies on the 5'UTR of the HCV replicons have revealed the importance of these regions on HCV genome replication. However, these studies did not distinguish their effects on either positive- or negative-strand RNA synthesis (Friebe et al., 2001; Kim et al., 2002b). The predicted secondary structures of positive- or negative-strand RNA of 5'UTR are slightly different (Schuster et al., 2002). In in vitro RNA synthesis using the full-length HCV RNA as the template, the NS5B polymerase is capable of positive-strand RNA synthesis, continuing from the 3' end of the full-length negative-strand RNA product, resulting in a dimeric hairpin HCV RNA (Oh et al., 1999). The significance of such a product is not clear.

THE HCV RNA REPLICATION MACHINERYHCV RNA replication is believed to occur in the cytoplasm of virus-infected cells based on the cytoplasmic localization of viral RNA (Gowans, 2000) and polymerase (Hwang et al., 1997; Selby et al., 1993). RNA is synthesized by a membrane-associated replication complex that includes the HCV RNA-dependent RNA polymerase (RdRP) NS5B, most of the other viral NS proteins (NS3, NS4A, NS4B, and NS5A), and possibly cellular proteins (Asabe et al., 1997; Bartenschlager et al., 1995; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Among the viral NS proteins, NS4B protein by itself induces membranous alterations that morphologically resemble the membranous webs found in replicon cells where viral RNA replication takes place (Egger et al., 2002; Gosert et al., 2003; Shi et al., 2003). A variety of biochemical evidence suggests that NS4B anchors the formation of the RNA replication complex (Gao et al., 2004), which is formed on the detergent-resistant membrane structures containing cholesterol-rich lipid rafts (Aizaki et al., 2004; Shi et al., 2003). Interestingly, all the nonstructural proteins, except NS5A, have to be translated in cis from the ORF of the very RNA molecule in order for RNA replication to occur (Appel et al., 2005). This finding suggests that the viral proteins are assembled in an ordered and sequential way into the replication complex soon after translation. The only trans-acting protein NS5A may enter the replication complex by binding to a cellular protein, VAP-33 (see below).

From the replicon studies, it appears that the RNA replication requires all the HCV nonstructural proteins except NS2. The NS3 is directly involved in RNA synthesis probably through its helicase function. The RNA helicase function is presumed to be necessary for unwinding the secondary structures of RNA template and to separate

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the positive- and negative-strand HCV RNA during replication. The HCV helicase lies within the C-terminal half of NS3, which has been shown to possess NTPase, single-stranded (ss) polynucleotide binding, and duplex-unwinding activities (Kim et al., 1995; Tai et al., 1996). NS3 alone has only a weak RNA unwinding activity, which can be significantly enhanced by the presence of NS4A (Pang et al., 2002). The resolution of the crystal structure of NS3 either alone or complexed with deoxyuridine octamer has provided additional insights into the mechanism of the HCV NS3 helicase function (Cho et al., 1998; Kim et al., 1998; Yao et al., 1999).

NS5B is a membrane-associated phosphoprotein (Hwang et al., 1997), which contains signature motifs, such as the GDD, shared by other viral RdRps (Koonin, 1991). The C-terminal 21 aa of NS5B plays a role in anchoring the protein to the membrane (Yamashita et al., 1998) but also plays a direct role in RNA synthesis (Lee et al., 2004b; Vo et al., 2004). NS5B also interacts with a SNARE-like cellular membrane protein, human vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (hVAP-33), which may directly or indirectly target the polymerase to the RNA replication site (Gao et al., 2004; Tu et al., 1999). Reduction of hVAP-33 expression either by dominant-negative mutants or small interfering RNA (siRNA) of hVAP-33 blocked the association of NS5B with detergent-resistant membranes and led to an inhibition of HCV RNA replication (Gao et al., 2004; Zhang et al., 2004).

Although multiple potential phosphorylation sites exist within the NS5B aa sequence, no site is conserved among all HCV isolates examined (Altschul et al., 1997), suggesting that phosphorylation of NS5B may vary among different isolates. Screening of a phage-display library with HCV NS5B protein as bait has identified one peptide with amino acid sequences homologous to protein kinase C-related kinase 2 (PRK2) (Kim et al., 2004b). In vitro analysis has revealed that PRK2 binds and phosphorylates the N-terminal region of NS5B. Further studies in the subgenomic replicon system have indicated that phosphorylation of NS5B by PRK2 is involved in the regulation of HCV RNA replication. It is not clear whether this phosphorylation has an effect on the NS5B polymerase activity and whether it is conserved among different isolates.

The crystal structure of NS5B shares significant similarity to those of other polymerases, but also displays certain striking differences (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). The domain organization in NS5B can be subdivided into the fingers, palm and thumb, similar to other polymerases. However, as other polymerases, such as the poliovirus 3D polymerase, are distinctly U-shaped, the fingers and the thumb domains of NS5B exhibit extensive contacts between each other, resulting in a globular-shaped molecule. The encircled active site is relatively inflexible and can accommodate only a template:primer duplex

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without global conformational changes. The C terminus of NS5B (excluding the hydrophobic tail) is present in the active site of the protein and has been hypothesized to play a role in the regulation of RdRp activity and template discrimination (Ago et al., 1999).

In in vitro RdRP assays, NS5B often uses the 3' end of the template RNA or an artificial oligonucleotide as a primer. (Al et al., 1998; Behrens et al., 1996; De Francesco et al., 1996; Ferrari et al., 1999; Lohmann et al., 1997; Yamashita et al., 1998; Yuan et al., 1997). However, it can also initiate de novo RNA synthesis in a primer-independent manner (Luo et al., 2000; Oh et al., 1999; Sun et al., 2000; Zhong et al., 2000). NS5B binds in vitro preferentially to several regions in the 3'-end of HCV RNA, including the 3'-coding region of NS5B, the U/UC-rich sequence, and part of the X region (in the stem I and II) (Fig. 1) (Cheng et al., 1999; Oh et al., 2000). Partial deletion of the 3'UTR of HCV RNA abolished the template activity of the RNA (Cheng et al., 1999; Oh et al., 2000). Thus, it appears that NS5B recognizes some specific sequence or structural elements at the 3' end of HCV RNA (Cheng et al., 1999; Oh et al., 2000). Once it binds the stem structure of the 3'UTR, however, NS5B initiates RNA synthesis only from the single-stranded RNA region closest to the 3' end of the template (Oh et al., 2000). This conclusion is supported by another study showing that the RdRp reaction mediated by NS5B requires a stable secondary structure and a single-stranded sequence with at least one 3'-end cytidylate in the RNA template (Kao et al., 2000).

Since the 3' end of HCV RNA ends with a near-perfect double-stranded stem (stem I) (Fig. 1), then how does HCV RNA synthesis initiate in vivo, if the in vitro mechanism reflects the mechanism of RNA synthesis in vivo? There are several potential mechanisms whereby the 3' end sequence of the viral RNA is retained during RNA replication: (1) The 3' end of HCV RNA may be extended by a terminal transferase so that there is a single-stranded tail at the 3' end to allow NS5B to initiate from the precise 3'-end. Indeed, an HCV cDNA clone containing two additional nt (UU) at the 3'-end of HCV RNA has been detected (Yamada et al., 1996). (2) RNA helicase or unwinding proteins may be present in the HCV replicative complex to unwind the 3'-end stem structure into the single-stranded region. (3) RNA synthesis may initiate internally in the single-stranded region within the 3'UTR; the 3'-end sequence may be recovered during the positive-strand RNA synthesis since the complementary sequence can be made by fold-back RNA synthesis. (4) The presence of other viral or cellular proteins may alter the choice of the initiation site of RNA replication.

HCV RdRp activity has been detected in the crude replication complexes prepared from lysates of cells carrying HCV replicons. This lysate can synthesize RNA from the endogenous template, but not exogenously added templates, and requires both

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NS5B and NS3 (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). The whole complex is localized on the detergent-resistant membrane and contains all the nonstructural proteins of HCV. The viral RNA is enclosed within the membrane complex and shielded from outside. All the nonstructural proteins are probably anchored on the membrane structures by a series of protein-protein interactions between them and with a cellular protein hVAP-33 (Tu et al., 1999). It has been shown that most of the HCV NS proteins, including NS3, NS4A, NS4B, NS5A, and NS5B, can interact with each other either directly or indirectly (Asabe et al., 1997; Bartenschlager et al., 1995; Gao et al., 2004; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Interestingly, while NS5B interacts with the N terminus of hVAP-33, NS5A binds the C terminus of hVAP-33. The importance of NS5A in HCV replication has been further suggested by the detection of a number of adaptive mutations clustered in a defined region of NS5A in a subgenomic HCV replicon (Blight et al., 2000). It is conceivable that this region may mediate the interaction of NS5A with a cellular protein that inhibits HCV replication. Further evidence supporting the existence of a replication complex consisting of multiple HCV NS proteins came from an analysis of the adaptive mutations derived from a subgenomic HCV replicon (Lohmann et al., 2001). An adaptive mutation in NS5B was found incompatible with those in NS5A or NS4B when introduced back into the same replicon. These mutations may affect contact sites between these proteins in the replication complex, resulting in a dramatic reduction in replication efficiency.

REGULATION OF HCV TRANSLATION AND REPLICATIONThe 5' and 3' UTRs are clearly the sites of important events leading to the onset of translation and replication of HCV RNA. The binding of viral or cellular proteins to the UTRs may modulate the secondary and/or tertiary structure of the viral RNA to facilitate its recognition by the translation machinery and/or the replicase complex. These proteins may recruit additional cellular factors and mediate long-range cross-talks between the ends of HCV RNA.

REGULATION OF TRANSLATION BY VIRAL AND CELLULAR PROTEINSThe HCV IRES-mediated translation is relatively inefficient as compared to that of other viruses (Borman et al., 1995). It has been suggested that HCV has a self-modulating mechanism to maintain a low level of replication and translation that may promote viral persistence. In this regard, it was speculated that domain IV of the IRES may be stabilized by interaction with the viral core protein, resulting in translation inhibition (Honda et al., 1996a). It has indeed been shown that the core protein binds to several sites within HCV IRES, thereby inhibiting translation (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). However, other studies have suggested that the core-coding sequence, rather than the core protein itself, is responsible for the suppression of IRES-mediated translation, possibly through long-range RNA-RNA interactions with the 5'UTR (Kim et al., 2003; Wang et al.,

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2000). The sites of RNA-RNA interaction have been mapped to nt 24-38 within the 5'UTR and nt 428-442 of the core-coding sequence (Kim et al., 2003), which is part of a stem-loop structure (Wang et al., 2000). The stem-loop IV of the IRES may be one of the candidates for feedback control, since the stabilization of this structure can reduce IRES activity and the primary sequence within this stem-loop is conserved in nearly all HCV strains (Honda et al., 1996a). However, these conflicting reports may have been due to the different reporter RNA constructs used in the different studies since the stable RNA structure assumed by some heterologous sequences fused directly at the initiation codon may be detrimental to translation directed by IRES (Rijnbrand et al., 2001). Furthermore, a cellular protein PTB binds to the 3'-end of the core-coding region and negatively regulates HCV translation (Ito and Lai, 1999). Thus, translation can be regulated by multiple RNA segments and viral proteins.

Several other HCV proteins, E2 (Taylor et al., 1999) and NS5A (Gale et al., 1997; He et al., 2003), may have an indirect effect on HCV translation by inhibiting PKR, but the biological significance of this effect is not clear.

Besides the canonical translation factors, such as the 40S ribosomal subunit and eIF3, the HCV IRES also recruits noncanonical cellular translation factors, such as La autoantigen (Ali and Siddiqui, 1997) and PTB (Ali and Siddiqui, 1995), which may regulate translation (Fig. 3). The La antigen is an RNA-binding protein belonging to the RNA recognition motif (RRM) superfamily (Gottlieb and Steitz, 1989). It has been implicated in various cellular processes (Ford et al., 2001; Gottlieb and Steitz, 1989) and the translation initiation of picornaviruses and flaviviruses (Ray and Das, 2002; Wolin and Cedervall, 2002). The La antigen recognizes the intact HCV IRES structure and significantly augments the IRES-directed translation in vitro (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004; Pudi et al., 2003; Pudi et al., 2004). Inhibition of HCV IRES activity caused by sequestration of La protein can be rescued by the addition of purified La protein (Das et al., 1998; Izumi et al., 2004). La protein binds to the GCAC motif near the initiator AUG within stem-loop IV (Pudi et al., 2003). Mutations in the GCAC, which alter the primary sequence while retaining the overall secondary structure, affect the binding of La protein to HCV IRES and significantly inhibit IRES-mediated translation both in vitro and in vivo (Pudi et al., 2004). It has been suggested that the nucleic acid-dependent ATPase activity of La may promote the transformation of stem-loop IV into single-stranded conformation, which is favorable for 40S ribosome binding and the formation of active initiation complex (Lemon and Honda, 1997; Pudi et al., 2004). In addition, La protein may enhance the binding of the ribosomal protein S5 to HCV IRES, which, in turn, facilitates the formation of the IRES-40S complex (Pudi et al., 2004). A recent study suggests that La antigen may also be involved in HCV RNA replication (Domitrovich et al., 2005).

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PTB interacts with three distinct pyrimidine-rich sequences within the HCV IRES (Ali and Siddiqui, 1995) (Fig. 3). The interaction of PTB with domain III of the IRES has been confirmed by electron microscopy analysis (Beales et al., 2001). Immunodepletion of PTB results in the loss of IRES-directed translation, which, however, cannot be restored by the addition of purified PTB, suggesting that additional factors tightly associated with PTB are also required to enhance IRES activity (Ali and Siddiqui, 1995). In addition to the IRES, PTB has also been shown to interact with the 3' X region (Ito and Lai, 1997; Tsuchihara et al., 1997) and to enhance HCV IRES-mediated translation (Ito et al., 1998). This long-range effect suggests that the HCV 5' and 3'UTR may interact with each other through PTB or other viral or cellular proteins. Furthermore, the presence of RNA aptamers of PTB inhibited HCV IRES translation (Anwar et al., 2000). In contrast, results obtained in a study of the subgenomic replicon system do not support a significant role of PTB in HCV replication (Tischendorf et al., 2004). However, PTB has been found in the detergent-resistant membrane complex in cells harboring the HCV subgenomic replicon, while it is in the detergent-sensitive membrane in

Fig. 3. Cellular proteins that interact with HCV RNA. The 5'UTR interacts with a basal translation factor (eIF3), noncanonical translation factors (PTB and La), and other cellular proteins that may regulate translation (hnRNP L and PCBP). The numbers in parentheses represent the nt sequence in the HCV genome, where the proteins bind. PTB has three distinct binding sites in the 5'UTR, whereas hnRNP L interacts with a region immediately downstream of the AUG codon. Both La autoantigen and PCBP recognize the entire 5'UTR. There is a PTB-binding site in the core-coding region, which plays a negative regulatory role in HCV translation. The 3'UTR is bound by a variety of proteins, all of which interact with the poly(U/UC) region. PTB also binds the X region. The length of poly(U/UC) affects the replication efficiency (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). These 5'UTR- and 3'UTR-binding proteins may affect viral replication (HuR, hnRNP C and GAPDH), translation (PTB), or RNA stability (La). VR, variable region.

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the control cells, indicating the recruitment of PTB to the HCV RNA replication complex; knockdown of PTB inhibited HCV RNA replication (Domitrovich et al., 2005)(Aizaki and Lai, unpublished).

Besides PTB, also known as heterogeneous nuclear ribonucleoprotein I (hnRNP I), several other proteins of the hnRNP family have been shown to interact with HCV IRES. hnRNP L specifically interact with the 3' border of the HCV IRES in the core-coding sequence; the binding correlates with the translation efficiency from the IRES (Hahm et al., 1998). The mouse minute virus nonstructural protein NS1-associated protein 1 (NSAP1) (Harris et al., 1999), a homolog of hnRNP R, also known as SYNCRIP (Synaptotagmin-binding cytoplasmic RNA-interacting protein) (Hassfeld et al., 1998), was recently shown to enhance IRES-dependent translation through the interaction with an adenosine-rich region in the 5'-proximal region of the core-coding sequence (Kim et al., 2004a; Reynolds et al., 1995). This protein appears to be involved in RNA replication as well (Choi and Lai, unpublished observation). Poly(rC)-binding protein (PCBP), which is also known as hnRNP E and involved in the expression regulation of numerous cellular and viral RNAs (Ostareck-Lederer et al., 1998), interacts with the HCV 5'UTR (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). PCBP has been implicated in the regulation of poliovirus IRES activity by binding to the 5'UTR of the viral genome (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). However, the specific interaction of PCBP-2 with the 5' terminal domain I of HCV RNA has no effect on IRES-mediated translation (Fukushi et al., 2001a). Consistent with the role of domain I in RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003), PCBP-2 may be involved in the replication rather than translation of HCV RNA.

Using a functional genomics approach, the proteasome α-subunit PSMA7 has been shown to be involved in IRES-mediated translation, but it is unknown whether the protein acts directly on IRES or indirectly through the regulation of other cellular proteins (Kruger et al., 2001). In summary, multiple cellular proteins binding to the 5' or 3'UTR can regulate HCV translation; some of them regulate both translation and RNA replication.

REGULATION OF RNA REPLICATION BY VIRAL AND CELLULAR PROTEINSAll the viral nonstructural proteins except NS2 are required for replication, but the modes of their participation are not clear. Adaptive mutations in the HCV replicons that allowed the replicons to enhance replication efficiencies have been detected in all of the viral NS proteins, particularly NS3 and NS5A, indicating that every viral nonstructural protein (except NS2) contributes to RNA replication. The purified recombinant HCV NS3 protein or its helicase domain alone can interact efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal

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RNA sequences (Banerjee and Dasgupta, 2001). Specific interaction of NS3 with the 3'-terminal sequences of the positive-strand RNA appears to require the entire 3'UTR. A predicted stem-loop structure present at the 3' terminus (nt 5 to 20 from the 3' end) of the negative-strand RNA, particularly the three G-C pairs within the stem, appears to be important for NS3 binding to the negative-strand UTR. This interaction may anchor RNA-protein complexes to the cytoplasmic membrane where viral replication complexes are formed.

The poly(U/UC)-rich region of the 3'UTR is a hot spot in the HCV genome for binding cellular proteins (Fig. 3), two of which are the Drosophila melanogaster embryonic lethal, abnormal visual system (ELAV)-like RNA-binding protein, HuR, and hnRNP C (Gontarek et al., 1999; Spångberg et al., 2000). Both HuR and hnRNP C interact with the 3' ends of both the positive- and negative-strand HCV RNA. Due to its pyrimidine-rich nature, it is not surprising that the poly(U/UC)-rich region has been identified to interact with PTB (Gontarek et al., 1999; Luo, 1999). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also interacts with the poly(U/UC) tract (Petrik et al., 1999), but the functional relevance of this interaction has yet to be determined. Based on studies of hepatitis A virus (HAV), the binding of GAPDH to the 5'UTR of HAV may directly influence IRES-dependent translation and/or replication of viral RNA by destabilizing the folded structure of the stem-loop IIIa of HAV IRES and competing with PTB for the binding to this structure (Schultz et al., 1996; Yi et al., 2000). The 3'UTR has also been shown to bind La autoantigen, which protects the HCV RNA from rapid degradation (Spångberg et al., 2001). Although the role of these proteins in HCV RNA replication has not be characterized, a group of host factors that bind to the 3'UTR of the closely related pestivirus BVDV has been shown to be required for viral RNA replication (Isken et al., 2003). It is conceivable that these cellular proteins are involved in not only RNA replication but translation as well, possibly through the 5' and 3' UTR interaction, causing the circularization of the viral RNA.

In addition to viral and cellular proteins, a liver-specific cellular microRNA, miR-122, was suggested by a recent study to regulate HCV RNA replication by directly interacting with the 5'UTR (Jopling et al., 2005). A 7-nt sequence (ACACUCC) complementary to the seed sequence of miR-122 was found in both the 5' and 3' UTRs and predicted to be potential binding sites for miR-122. Disruption of sequence complementarity between the 5'UTR, but not the 3'UTR, and miR-122 reduced HCV RNA replication without affecting RNA stability or translation. It is speculated that miR-122 may aid in RNA folding or RNA sequestration in replication complexes. Since miR-122 is expressed in Huh7 but not HepG2 cells, it may also play a role in host-range determination.

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THE EVIDENCE FOR DIRECT OR INDIRECT 5'-3' INTERACTIONTranslation of vast majority of eukaryotic mRNAs, which are capped at the 5' end and polyadenylated at the 3' end, has been shown to adopt a closed-loop mechanism, in which the mRNAs are circularized via a 5'-3' interaction mediated by the cap-binding proteins, eIF4F and eIF4G, and the poly(A)-binding protein, PABP. The eIF4G-PABP interaction has also been shown to be required for poly(A)-mediated stimulation of picornaviral IRES-dependent translation, indicating that the 5'-3' crosstalk is mechanistically conserved between classical eukaryotic mRNAs and picornaviral RNA (Herold and Andino, 2001; Michel et al., 2001; Michel et al., 2000). Circularization has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). Even in the absence of a poly(A) tail in HCV RNA, the closed-loop model may still be preserved in HCV IRES-mediated translation by the presence of RNA sequences and proteins that can functionally replace the poly(A) tail and PABP (Ito and Lai, 1999). Indeed, the X region of the 3'UTR has been shown to bind PTB and enhance translation of HCV RNA (Ito et al., 1998), suggesting that the functions of the X region may be similar to that of poly(A) in eukaryotic mRNA translation (Kahvejian et al., 2001). Since PTB also interacts with the 5'UTR, it may mediate crosstalk between the 5'- and 3'-ends of HCV RNA. Thus, the mechanism of translation enhancement by PTB may be similar to that of eIF4G-PABP in the translation of cellular and viral RNAs that contain a poly(A) tail.

SWITCH BETWEEN TRANSLATION AND RNA REPLICATIONSince RNA replication and translation occur on the same RNA molecules, the question arises how these two processes are coordinated. For some RNA viruses, there is evidence of coupling between RNA replication and translation. For example, poliovirus defective-interfering RNA without a translatable ORF can not replicate; the nature of the protein product is not critical, but the translatability is essential (Collis et al., 1992; Hagino-Yamagishi and Nomoto, 1989; Novak and Kirkegaard, 1994). This requirement has been demonstrated for several other viral RNAs, such as clover yellow mosaic virus RNA (White et al., 1992), Kunjin virus (Khromykh et al., 2000), and rubella virus (Liang and Gillam, 2001). In coronavirus, the cis-acting protein appears to confer a replication advantage to the RNA; the longer the ORF, the more robust the RNA replication is (de Groot et al., 1992; Kim et al., 1993; Liao and Lai, 1995). The mechanism of the coupling of these two processes is not yet clear. However, there are also viral RNAs (e.g., vesicular stomatitis virus, influenza virus, Sindbis virus) whose replication does not depend on translation of the ORF on the same RNA. In any case, translation and replication must be separated since translation goes in the 5' to 3' direction, whereas negative-strand RNA synthesis goes from 3' to 5' on the same positive-strand RNA template. When the translation machinery meets the replication complex in opposite direction, there must be a mechanism to prevent confrontation. The situation is akin to the separation of transcription and replication of cellular DNA.

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In HCV, the 5' and 3'UTR sequences are involved in the regulation of both translation and RNA replication. There is substantial overlap in the UTR regions required for translation and RNA replication. Nevertheless, the structural and sequence requirement for these two processes may be different. It is conceivable that the structural changes involved in translation and RNA replication may be effected by the viral or cellular proteins binding to these regions. Indeed, several cellular proteins binding to the 5' and 3'UTR of HCV have been shown to affect both translation and replication.

In poliovirus, a switch between translation and RNA replication has been proposed to be controlled by PCBP, which enhances translation by binding to the 5'-terminal cloverleaf structure of the poliovirus RNA, and the viral 3CD polymerase, which promotes negative-strand RNA synthesis by binding to the same RNA structure, possibly by altering the structure of this region (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). Interestingly, PCBP-1 and 2 have also been shown to interact with the HCV 5'UTR, with PCBP-2 binding particularly to stem-loop I, suggesting a possibly similar role of these proteins in regulating a switch between HCV RNA replication and translation (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). In addition, the HCV core protein may also be involved in the switch by down-regulating IRES-dependent translation as a regulatory mechanism required for the initiation of RNA replication (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). Since many of the cellular proteins binding to the 5' and 3'UTR of HCV have been reported to regulate both translation and replication, it is conceivable that the relative ratios of the different proteins may control the switch between translation and replication. Furthermore, the HCV RNA elements required for translation and those for replication partially overlap. So, the key question in this regard is how the structures of these elements are altered by RNA-RNA or protein-RNA interactions so that the RNA can be properly directed to be used for translation or replication.

Alternatively, an entirely different mechanism may operate to regulate translation and RNA replication of HCV. The RNA replication complex has been shown to reside in the cholesterol-rich, detergent-resistant membrane complex (Aizaki et al., 2004; Shi et al., 2003), whereas translation occurs on the detergent-sensitive, endoplasmic reticulum membrane. Thus, there may be separate machineries in different subcellular compartments for these two processes. The different viral and cellular proteins may bind to RNA molecules differentially in these two different compartments. The key question in this regard is how the RNA is transported from the replication complex to the site of translation or vice versa so that these two functions can be separated.

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PERSPECTIVESThe 5' and 3'UTR are the most conserved regions of HCV RNA and play key roles in regulating translation and RNA replication. The knowledge on these two processes is still rudimentary, but the development of subgenomic and genomic replicons and the infectious culture systems (Lohmann et al., 1999; Wakita et al., 2005) provides promises for the unraveling of these two processes in the near future. These two regions also offer promising targets for developing antiviral agents.

Within the past two years, small molecule inhibitors of the NS3 protease and the RNA-dependent RNA polymerase have been shown in early clinical studies to be efficacious in both treatment-naïve patients and patients who failed interferon therapy. However, the extensive genetic heterogeneity of HCV RNA and the rapid evolution of quasispecies present a substantial challenge for these inhibitors to broad-spectrum activity. The high degree of sequence conservation in the 5'UTR and 3'UTR among different HCV genotypes makes these regions attractive targets for antiviral therapies, such as antisense oligonucleotides (Soler et al., 2004), ribozymes (Welch et al., 1996; Welch et al., 1998), and siRNAs (Kronke et al., 2004; Randall and Rice, 2004). The inhibition of HCV RNA translation or replication has been observed with these inhibitors that target the 5'UTR alone or together with the core-coding sequence of HCV (Hanecak et al., 1996; Kronke et al., 2004; Macejak et al., 2000; McCaffrey et al., 2003; Ohkawa et al., 1997; Sakamoto et al., 1996). Universal siRNAs targeting similar regions have been generated and proven to be effective against all known genotypes (Kronke et al., 2004; Yokota et al., 2003). Encouragingly, early clinical trials have demonstrated efficacy of some of these inhibitors in HCV-infected patients despite the limitations associated with RNA-based therapies and the inherent structures of the UTR sequences (Branch, 1998; Crooke and Bennett, 1996; Gomez et al., 2004). The interventions directing against conserved domains of viral RNAs may provide valuable alternatives to small molecule inhibitors that target HCV proteins.

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The HCV Core Protein

Chapter 3

Assemble and Interact: Pleiotropic Functions of the HCV Core Protein

Stephen J. Polyak, Kevin C. Klein, Ikuo Shoji, Tatsuo Miyamura and Jaisri R. Lingappa

ABSTRACTWhile surrogate capsid assembly model systems are currently the best tools for studying HCV core assembly, bona fide HCV culture systems are being developed. The time will soon come when HCV culture systems and small animal models will be the norm, rather than the exception (see Chapters 12 and 16). It is now clear that HCV core protein interacts with many cellular proteins and signal transduction pathways, that HCV quasispecies influence biologic responses, and HCV proteins such as core can have different effects depending on whether the protein is encountered inside or outside the cell. The studies discussed herein have enhanced the understanding of HCV capsid assembly and the role(s) of HCV core and host cell interactions in the establishment of persistent infection and the pathogenesis of HCV liver disease. Continued studies of this nature will also provide a basis for the rational design of vaccines and novel therapeutics against HCV infection in humans.

INTRODUCTIONAs covered elsewhere in this book, HCV infection is a serious global health problem, which accounts for billions of dollars in medical expenses in the US alone (Kim, 2002). Clinically, acute HCV infection is frequently anicteric and asymptomatic. The situation is compounded given the natural tendency for acute HCV infection to progress to chronic infection. Thus, more effective strategies to successfully cure patients of their infection are urgently needed. This chapter focuses on a key HCV molecule, the HCV core or nucleocapsid protein.

THE CORE OF THE PROBLEMThe HCV core protein has been reported to have many functions. With respect to the virus, the main function of the core protein is to form the capsid shell that will house and protect the HCV genomic RNA while the virus passes from one cell to another, or from one person to another. However, the HCV core protein also modulates many different host pathways by interacting with a variety of cellular factors. In the following sections, we will highlight important new developments in HCV capsid assembly and HCV core-host interactions.

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THE ROLE OF HCV CORE IN CAPSID ASSEMBLY

WHAT IS A CAPSID?A viral capsid is the protein shell that encapsidates and protects the viral genome. Viral capsids can be composed of one or more virus-encoded proteins. In the case of enveloped viruses, after assembling and encapsidating the genomic RNA, a viral capsid then facilitates virion formation by interacting with the viral envelope glycoproteins and budding. The budding process is sometimes, but not always, mediated by the viral capsid. For example, the capsid proteins of Ebola and HIV contain domains that regulate budding, while in the case of tick borne encephalitis (TBE) virus, it is the envelope glycoproteins that mediate budding. These events (capsid assembly, encapsidation, and budding) are typically referred to as late events in the viral life cycle. For HCV, as will be discussed below, many details of the late events in the HCV life cycle are unclear.

In the case of HCV, as is true for all members of the Flaviviridae, the core protein is the only viral protein present in the capsid. The final nucleocapsid contains genomic RNA, coated and protected by the capsid. HCV, being an enveloped virus, has a lipid envelope, containing the viral envelope glycoproteins as well as host membrane proteins, surrounding the nucleocapsid. The late events of the HCV life cycle, including capsid and virion assembly, are shown schematically in Fig. 1. In this section, we will focus on HCV core, its characteristics, what is known about its assembly into a bona fide HCV capsid, and the blocks to HCV capsid assembly that exist in mammalian cell culture systems.

PROPERTIES OF HCV COREThe HCV genomic RNA is approximately 9.6 kilobases in length and encodes a single, large polyprotein of about 3000 amino acids (aa). The polyprotein is cleaved by viral and cellular proteases to generate at least 10 viral proteins (Suzuki et al., 1999). The core protein represents the first protein in the polyprotein, followed by two glycoproteins, E1 and E2. The immature form of HCV core contains 191 aa. These 191 aa have been separated into three general domains (McLauchlan, 2000). The first domain (domain I), encompassing aa 1 - ~122, is highly basic and very hydrophilic. This domain is thought to be responsible for binding RNA and mediating capsid assembly, and has been reported to interact with many cellular proteins. The second domain encompasses the majority of the C-terminus of HCV core. In contrast to domain I, domain II is hydrophobic. Thus, domain II mediates interactions with lipids and membrane proteins and is not present in capsid proteins of most other viruses in the Flaviviridae. The final domain (III), which is very hydrophobic and is predicted to form an alpha helix, is at the extreme C-terminus of the immature core protein, and corresponds to the signal sequence for E1 (aa 175 - 191). This domain is cleaved soon after core is translated and is absent from the

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mature form of HCV core. Nevertheless, domain III appears to be very important in terms of HCV core stability, targeting, and function. Two major forms of core protein, corresponding to 21- and 23-kDa (p21 and p23), are generated in vitro and in cultured cells (Yasui et al., 1998), corresponding to the mature (signal cleaved) and immature (signal uncleaved) forms of the protein.

HCV CORE BIOGENESISSynthesis of HCV core in the same polyprotein as the HCV envelope proteins creates an interesting predicament in that core, the capsid protein, needs to be soluble and cytoplasmic, while the envelope glycoproteins are transmembrane and anchored into the host membrane. Therefore, like other flaviviruses, HCV has evolved an internal signal sequence for E1, the first envelope glycoprotein (referred to as E1 SS or domain III, as described above). The E1SS is encoded between HCV core and E1. Thus, after core is translated, the nascent polyprotein is targeted to the ER translocation channel by the E1 SS (Fig. 1). A host enzyme located in the ER, signal peptidase, cleaves just proximal to the E1 SS, releasing the immature form of core from the polypeptide (Hijikata et al., 1991; Santolini et al., 1994). A different endoplasmic reticulum (ER) enzyme, signal peptide peptidase (SPP), subsequently cleaves just before the E1 SS liberating the mature form of HCV core at the cytoplasmic face of the ER (McLauchlan, 2000; McLauchlan et al., 2002). SPP is a presenilin-type aspartic protease that catalyses intramembrane proteolysis of signal sequences and membrane proteins within the ER (Weihofen et al., 2002). Precise mutational analyses have shown that intramembrane cleavage by SPP is abolished when helix-breaking and -bending residues in the C-terminal signal sequence are replaced by basic residues. Furthermore, the signal sequence itself and three hydrophobic aa Leu-139, Val-140, and Leu-144 of the core protein

Fig. 1. Overview of capsid/virion assembly. Genomic RNA is translated by a host ribosome. HCV core is the first polypeptide encoded in the polyprotein. Just proximal to core is the membrane envelope glycoprotein E1. The signal sequence (SS) for E1 (distal to core) targets the polyprotein to the ER. Signal peptidase cleaves the immature form of core from the growing polypeptide. Signal peptide peptidase then cleaves the E1 SS releasing the mature form of core. Core then multimerizes and encapsidates HCV RNA at the cytoplasmic face of the ER. Capsids that are formed in the cytoplasm then interact with E1 and bud into the ER lumen. Enveloped virions are then released, presumably via the secretory pathway.

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are required for SPP cleavage, although none of these residues are essential for cleavage at the core-E1 junction by signal peptidase, or for translocation of E1 into the ER (Okamoto et al., 2004). The exact cleavage site for producing mature core (p21) is still controversial, since Leu-179 (Hussy et al., 1996; McLauchlan et al., 2002), Leu-182 (Hussy et al., 1996), Ser-173 (Santolini et al., 1994), and Phe-177 (Okamoto et al., 2004) have all been reported as potential sites of cleavage. After being cleaved into the mature form at the ER, core can undergo a number of possible fates, including assembly into capsids, targeting to other organelles, and interaction with host proteins resulting in modulation of various cellular processes, as will be discussed in more detail below.

HCV CAPSID STRUCTURE AND PROPERTIESThe main role for HCV core in the viral life cycle is to form a nucleocapsid to protect the viral genome. Once cleaved from the polyprotein, the mature core protein presumably assembles into HCV capsids, most likely at the cytoplasmic face of the ER (Mizuno, 1995; Blanchard, 2002; Blanchard, 2003). Unfortunately, no cellular system robustly recapitulates late events in the viral life cycle, although there may be hope with the recent development of an infectious HCV system (see Chapter 16). For this reason, mechanistic details of this process are lacking. HCV replicon systems (see Chapter 11), first developed in 1999, represented a major breakthrough because they allowed replication of HCV RNA in mammalian cells (Blight et al., 2000; Lohmann et al., 1999). However, even when HCV core is synthesized to high levels, late events in the HCV life cycle do not occur in most replicon systems, as judged by electron microscopy (Pietschmann et al., 2002). Therefore a number of model systems have been developed to study the structure of HCV capsids and HCV capsid assembly.

Knowledge of HCV capsid appearance in vivo has come from examining particles in serum or in infected liver biopsies. Non-enveloped capsids have been observed in the cytoplasm of liver cells, while enveloped particles have been seen in the cisternae of the ER, as judged by transmission electron microscopy (TEM) (Bosman et al., 1998; Shimizu et al., 1996). The presence of capsids at or in the ER by TEM in numerous studies implicates the ER as the site of HCV capsid assembly (Blanchard, 2002; Maillard, 2001; Mizuno, 1995; Shimizu, 1996). More recently, a careful TEM analysis of HCV virions and non-enveloped nucleocapsids from serum of HCV infected patients was performed (Maillard et al., 2001). This study revealed that non-enveloped HCV nucleocapids can be found in significant quantities in serum. These capsids, as well as those obtained by detergent treatment of enveloped virions, are spherical but heterogeneous in size, with a bimodal distribution of capsid diameters corresponding to ~38 - 43 nm and ~54 – 62 nm. It remains unclear what governs capsid size and whether the size differences are biologically significant. Unfortunately, unlike with other flaviviruses, visualization of HCV virions or capsids at atomic resolution has not yet been achieved.

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Biochemical analyses have determined that enveloped HCV virions have a density 1.08 to 1.16 g/ml (Bradley et al., 1991; Kaito et al., 1994; Kanto et al., 1994; Miyamoto et al., 1992). Similar studies on non-enveloped HCV capsids have yielded conflicting results. HCV capsids with envelopes removed using detergent have densities of approximately 1.25 g/ml (Kaito et al., 1994; Kanto et al., 1994; Miyamoto et al., 1992) or 1.32 - 1.34 g/ml (Maillard et al., 2001; Shindo et al., 1994), with the electron microscopic appearance of capsids of both densities being otherwise very similar (Maillard et al., 2001). An explanation has been proposed to explain the finding of two different buoyant densities: capsids that band at the lower density (~1.25 g/ml) appear to be associated with fragments of membranes, while those banding at the higher density (~1.32 g/ml) appear to be free of membranes (Maillard et al., 2001). However, this hypothesis remains to be tested. Additionally, it appears that both the immature and mature form of core can assemble and be incorporated into capsids, although, not surprisingly, the mature form is the main species in virions (Yasui et al., 1998).

MODEL SYSTEMS FOR CAPSID ASSEMBLYWhile electron micrographs of infected serum and hepatocytes give a literal snapshot of what is occurring in vivo, at the other extreme are minimal systems that can be used as surrogates for understanding the process of capsid assembly. In these minimal systems, purified recombinant core is incubated with RNA in the absence of other cellular factors. In the presence of RNAs containing a high degree of secondary structure (e.g. tRNA or the HCV 5' untranslated region), C-terminal truncation mutants were found to assemble into regularly shaped capsids that resemble HCV capsids from infected individuals (Kunkel et al., 2001). Similar results were obtained by expressing truncated core constructs in E. coli (Lorenzo et al., 2001). In contrast, full-length (wild-type) recombinant core assembles into particles with irregular shapes (Kunkel et al., 2001), raising the possibility that host factors or co-ordination of assembly with core synthesis may be required to assemble proper capsids from full-length HCV core. These studies also demonstrated that domain I is sufficient for core assembly. Furthermore, removal of domain II appeared to facilitate capsid assembly, allowing the purified core protein to assemble into more regular-shaped capsids. Together, these systems show that HCV core contains all of the information to assemble into capsid-like structures (in the presence of RNA) (Kunkel and Watowich, 2002). However, because of the minimalist nature of these systems, other systems will be required to determine the mechanism by which wild-type core assembles into capsids within cells, where assembly is likely to be influenced by other events including de novo core translation, host factors, and targeting of core to specific organelles.

A cell-free system, a virtual hybrid between in vitro systems and cellular systems, has recently been developed to study HCV assembly. In these systems, cellular

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extracts are used to reconstitute and link translation to post-translational events, such as capsid assembly. Thus, these systems combine the benefits of being able to manipulate the assembly reaction in a test tube while maintaining a cellular context. Cell-free systems faithfully reconstituted HCV capsid assembly when full-length core, either the immature or mature form, was expressed de novo in either wheat germ extracts or rabbit reticulocyte lysate (Klein et al., 2004). Moreover, TEM analysis revealed that capsids formed from full-length core in the cell-free system were morphologically very similar to capsids produced in infected patient serum, both in size and structure (Klein et al., 2004), thereby validating the cell-free system for mechanistic and mutational studies. In addition, cell-free HCV capsid assembly is very efficient, with over 60% of newly-synthesized core polypeptides assembling into immature capsids (Klein et al., 2004).

Some cellular systems have also been used to study capsid assembly. When over-expressed in insect cells, core assembles into 30 – 60 nm particles at the ER (Baumert et al., 1998; Baumert et al., 1999; Maillard et al., 2001) that closely resemble capsids produced in vivo. When the envelope proteins E1 and E2 are also expressed, capsids can be seen budding into the ER and cytoplasmic vesicles (Baumert et al., 1998); however, unfortunately no virus-like particles are released (Baumert et al., 1998; Baumert et al., 1999; Maillard et al., 2001). Therefore, this system recapitulates much of what is seen in hepatocytes and supports the notion that capsids assemble at the ER, although virion production is still blocked at a later step in the viral life cycle. Nucleocapsid-like particles have also been observed upon expression of HCV core in yeast (Majeau, 2004).

In contrast to these model systems, in general, mammalian cell lines do not support HCV capsid assembly. There have been isolated reports of capsids being produced in cultured mammalian cells (Blanchard, 2002; Ezelle, 20026; Mizuno, 1995); however, the extent of HCV assembly in these cells is unclear. As noted above, even in replicon cells with high levels of HCV core synthesis, HCV assembly is not supported (Pietschmann et al., 2002; Bukh et al., 2002), similar to most cultured mammalian cells (Hope and McLauchlan, 2000). These findings suggest that mammalian cell lines either lack a necessary cellular factor(s) or contain inhibitory factor(s) that cause the majority of core to be targeted away from the ER, as discussed below. This alternate localization of core (Pietschmann et al., 2002), possibly in conjunction with other negative regulatory influences, correlates with failure to assemble HCV capsids or virions in cultured cell lines. Consistent with this, when crude hepatocyte extracts containing membrane-bound organelles are added to the highly permissive cell-free capsid assembly system, efficiency of assembly is reduced (Klein et al., 2004).

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HCV ASSEMBLY: REQUIREMENTS AND MECHANISTIC ANALYSISAlthough an ideal model system for HCV capsid assembly does not exist, much has been elucidated about the requirements and process of capsid assembly from the various systems mentioned above. In vitro studies have been useful for structural analyses, having revealed that HCV core undergoes a conformational change upon assembling into capsid like structures (Kunkel and Watowich, 2002). Meanwhile, the cell-free system for HCV capsid assembly has allowed the process of core assembly to be analyzed mechanistically (Klein et al., 2005; Klein et al., 2004). Pulse chase analyses in the cell-free system have revealed that assembly occurs very quickly, with very little delay between completion of translation and completion of assembly (Klein et al., 2004). Additionally, capsid assembly was not highly dependent on protein concentration or membranes, unlike many other viruses. When HCV core expression was decreased 200 fold, only a 2.3 fold decrease in amount of assembly was observed (Klein et al., 2004). Both the speed of HCV capsid assembly and its relative concentration independence differ from what has been seen with assembly of other types of viral capsids (i.e. lentiviruses and hepadnaviruses) in analogous cell-free systems (Lingappa et al., 1997; Lingappa et al., 1994; Lingappa et al., 2005). Thus, the basic assembly mechanism of HCV capsids may differ from that of many other viral capsids that assemble at the cytoplasmic face of membranes. Assembly may occur in microenvironments, for example on polysomes that contain a high concentration of core protein translating off a single mRNA. The presence of high local concentrations of newly-synthesized HCV core polypeptides, possibly in conjunction with cellular factors, could promote rapid and efficient HCV assembly in permissive cellular extracts, although future studies will be required to test this hypothesis.

Model systems for HCV assembly have also been used to define regions of HCV core that are important for HCV capsid assembly. Studies using recombinant HCV core truncation mutants have revealed that domains II and III are dispensable for assembly (Kunkel et al., 2001; Lorenzo et al., 2001). In fact, truncation mutants lacking these domains assemble better than full-length constructs in vitro (Kunkel et al., 2001). Systematic analysis of HCV capsid truncation, deletion, and point mutants in the cell-free HCV capsid assembly system have confirmed that the C-terminus is dispensable for assembly, and also demonstrated that the N-terminal 68 aa are required for capsid assembly (Klein et al., 2005; Klein et al., 2004). This region of HCV core contains numerous basic residues organized into two clusters. Removing either cluster of basic residues, or mutating as few as 4 basic residues to alanines in either cluster, significantly reduces assembly of capsids in wheat germ extracts (Klein et al., 2005). Conversely, when neutral aa were deleted from the same region, no effect on cell-free HCV capsid assembly was observed, suggesting that the critical determinant for assembly is the overall basic charge of the N-terminus. Likewise, deletions or mutations in other regions of HCV core

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did not affect assembly (Klein et al., 2005). While these studies indicate that basic residues in the N-terminus are critical for assembly, it remains unclear whether the N-terminal 68 residues are sufficient for assembly. It should be noted that other domains of core are clearly important for interaction of core with cellular factors and for trafficking of HCV core to distinct cellular locations, as discussed below. Domains involved in core trafficking and cellular protein interactions are likely to influence or even regulate HCV capsid assembly in intact cells, but these events have not been studied together due to lack of cell lines that recapitulate HCV capsid assembly in a robust manner.

RNA BINDING AND ENCAPSIDATION BY COREBesides multimerization to form the capsid, the other major function performed by core during assembly is RNA encapsidation. Many viruses will encapsidate non-specific cellular RNA if viral genomic RNA is not present. Moreover, many viruses use RNA as a scaffold for assembly, and/or to nucleate the assembly process. HCV core appears to act similarly. Domain I of HCV core is extremely hydrophilic, largely due to the many basic residues clustered in this region. Basic residues are frequently involved in nucleic acid binding because the positive charge can interact with the negative phosphate backbone of nucleic acids. Indeed, HCV core binds RNA (Fan et al., 1999; Santolini et al., 1994; Shimoike et al., 1999) and this association is dependent on the basic N-terminus (Santolini et al., 1994). Consistent with this, and supporting the notion that RNA acts as a scaffold for assembly, RNA was required for in vitro assembly (Kunkel et al., 2001). Additionally HCV core has RNA chaperone capabilities, suggesting that core may also help restructure RNA, which may have implications for specific genomic encapsidation (Cristofari et al., 2004).

While the notion that HCV core binds to RNA is well established, it is unclear whether HCV core preferentially binds HCV genomic RNA over cellular RNAs. Core has been shown to bind ribosomal RNA (Santolini et al., 1994), tRNA (Kunkel et al., 2001), and HCV genomic RNA (Cristofari et al., 2004; Fan et al., 1999; Kunkel et al., 2001; Shimoike et al., 1999). It appears that the only requirement is that the RNA should contain significant amounts of secondary structure. When recombinant core was incubated with denatured, or unstructured, RNA, it failed to assemble into capsids suggesting that it could not interact with unstructured RNA. Conversely, when highly structured tRNA or the HCV UTR was used, core assembly was promoted (Kunkel et al., 2001).

If core binds to any structured RNA, how does genomic RNA get specifically packaged? Many viral capsid proteins have a higher affinity for specific structures in their cognate genomic RNA, allowing them to preferentially bind the proper RNA. It is unclear whether HCV core has higher affinity for HCV genomic RNA. One

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study demonstrated that the HCV core protein binds specifically to a radiolabeled probe containing the 5' UTR of the genomic RNA. This interaction was abolished by excess unlabeled probe, but not by unlabeled, non-specific RNA, suggesting that core preferentially binds genomic RNA (Fan et al., 1999). This could explain how genomic RNA gets selectively packaged into virions over other cellular RNAs. Conversely, Santolini et al. reported that core fusion proteins bind equally well to HCV genomic RNA and heterologous RNA, suggesting that HCV core does not have enough specificity in its binding to promote genomic RNA encapsidation (Santolini et al., 1994). If HCV core does not specifically bind genomic RNA, then some other mechanism must exist to promote encapsidation of the genome. One possibility is that assembly occurs in microenvironments that contain only a single species of mRNA (i.e. HCV genomic RNA), as discussed above. Unfortunately, RNA encapsidation has not yet been analyzed in conjunction with capsid assembly in any system, so it remains unclear exactly what RNAs are encapsidated and how HCV core selects RNA for encapsidation during synthesis and assembly.

CAPSID ASSEMBLY: LIGHT AT THE END OF THE TUNNEL?As mentioned, for the most part cell culture systems do not support virion production, or even capsid assembly. However, isolated reports have identified infectious virus propagated in special cell culture systems and at low levels. One group infected hepatocytes that were cultured in a radial-flow bioreactor and found that HCV is able to replicate to very low titers (Aizaki et al., 2003). Additionally, at the 11th International Meeting on Hepatitis C and Related Viruses in Heidelberg in October 2004, there were three reports of very low titer infectious virus particle formation in cells transfected with HCV genomic RNA (Murakami et al., 2004b; Pietschmann et al., 2004; Wakita et al., 2004). These initial studies have been confirmed by independent groups (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) (See Chapter 16). Use of 3-dimensional cultures (Murakami et al., 2004b) or transfection with the JFH strain (Pietschmann et al., 2004; Wakita et al., 2004) resulted in production of infectious particles. In one case infection was receptor mediated, as antibodies to the putative HCV receptor, CD81, blocked infection (Pietschmann et al., 2004). Unfortunately, in all cases, levels of virus production were too low to result in measurable titers or any ultrastructural evidence of virus formation (Aizaki et al., 2003; Murakami et al., 2004b; Pietschmann et al., 2004; Wakita et al., 2004). Most recently, Heller et al. also isolated virus like particles from cell culture after transfecting RNA corresponding to the exact genomic sequence (Heller et al., 2005). This study also showed morphologic data, which suggests that the particles produced are, indeed, virions. Thus, a new wave of data shows evidence that HCV can assemble into capsids and, subsequently, into virions in mammalian cell culture. However, it is unclear whether assembly in these systems is just a stochastic event, what amount of virus or virus like particles are produced, how assembly is regulated in these systems, or what cellular subpopulation, if

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any, is producing the limited number of viruses. By using a combination of all of the current model systems, as well as, newly described cellular systems, new insights into the mechanism by which HCV assembly is regulated in cells should be elucidated. This could allow for enhancements of current cell culture systems that could in turn facilitate the study of late events of the HCV life cycle in cells.

SUB-CELLULAR TARGETING OF HCV CORE

IF CORE DOES NOT ASSEMBLE, WHERE DOES IT GO?While much information has been elucidated from the various model systems outlined above, surprisingly, mammalian cell lines including human liver-derived cell lines fail to produce quantifiable levels of HCV capsids, or virions. For reasons that remain unclear, in these cell lines HCV core polypeptides can be directed to alternate cellular locations upon release from the nascent HCV polyprotein. The fact that core assembles efficiently in infected humans and chimpanzees, but not in intact cultured cell-lines, suggests that HCV assembly can be negatively regulated. Trafficking of core to alternate sites is one possible mechanism for negative regulation of capsid assembly.

One approach to studying the subcellular localization of core involves immunostaining liver biopsy specimens from infected patients. This has revealed that the core protein predominantly localizes within the cytoplasm of infected hepatocytes, and often shows a punctate granular distribution within cells (Gonzalez-Peralta et al., 1994; Gowans, 2000; Sansonno et al., 2004; Yap et al., 1994). However, when the core protein alone or the entire viral polyprotein are expressed in mammalian cells, the majority of core has been observed at the ER membrane (Lo et al., 1995), on the surface of lipid droplets (Barba et al., 1997; Hope et al., 2002; McLauchlan et al., 2002; Pietschmann et al., 2002; Shi et al., 2002), and on mitochondrial and mitochondrial-associated membranes (Schwer et al., 2004; Suzuki et al., 2005). In addition, core is also known to target to the nucleus (Lo et al., 1995; Matsuura et al., 1994; Moriishi et al., 2003; Moriya et al., 1998; Yasui et al., 1998), where it can be a substrate for proteasomal degradation.

What governs whether core stays at the ER to assemble or traffics to other areas of the cell is not completely understood. Nevertheless, it is clear that such regulation exists and is quite complex. The finding that core targets to lipid droplets and mitochondria, but E1 and E2 do not (Pietschmann et al., 2002; Schwer et al., 2004), raises the possibility that targeting of core away from the ER occurs at a very early time after core synthesis, before core has had time to interact with the envelope glycoproteins. Furthermore, a number of studies suggest that aa in domains II and III direct the post-translational trafficking of core, although agreement is lacking as to which residues are critical. Okamoto et al. has shown that not only the C-

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terminal signal sequence but also aa 128-151 are required for ER retention of the core protein by using a series of N-terminally truncated core protein constructs (Okamoto et al., 2004). Suzuki et al. has reported that a region of aa 112-152 mediates association of the core protein with the ER in the absence of the C-terminal signal sequence (Suzuki et al., 2005). McLauchlan et al. have proposed that a large part of the core protein remains within the cytoplasmic leaflet of the ER membrane after SPP cleavage (McLauchlan et al., 2002). Upon intramembrane cleavage of the transmembrane signal peptide, the processed core protein may traffic along the lipid bilayer from the site of biosynthesis to zones at the ER, where lipid droplets are produced (McLauchlan et al., 2002).

Deletion analyses have revealed that domain II (in particular residues between aa 125 - 144) plays a critical role in targeting core to lipid droplets (Hope and McLauchlan, 2000; Hope et al., 2002). Notably, no domain homologous to domain II is present in the core proteins of related pesti- and flavi-viruses. In contrast, the core protein of GB Virus B, from the GB virus group within the Flaviviridae, does contain a homologous domain that also appears to mediate targeting to lipid droplets (Hope et al., 2002). Domain II contains two closely spaced prolines that form a proline knot and appear to be required for targeting core to lipid droplets. The region containing this proline knot can be replaced with a proline knot domain from lipid-associated plant proteins called oleosins (Hope et al., 2002), with preservation of lipid targeting. Lipid targeting of HCV core can also be altered by mutations that affect SPP cleavage. Helix-breaking point mutations within the signal sequence (domain III) eliminate SPP cleavage, but also eliminate trafficking to lipid droplets, leaving core protein on the cytoplasmic face of the ER (McLauchlan et al., 2002; Okamoto et al., 2004). While these alternate pathways for core trafficking are beginning to be defined, the downstream consequences of different post-translational trafficking pathways on core function have not yet been explored. This is in part because using core mutants to study these cellular fates has proven to be relatively tricky. Studies have shown that C-terminally truncated versions of the core protein are localized exclusively to the nucleus (Suzuki et al., 1995). A fraction of the core protein was detected in the nucleus even when full-length HCV core gene was expressed, suggesting that the mature core protein also localizes to the nucleus (Moriya et al., 1997a; Yasui et al., 1998). The N-terminal domain of the core protein contains three stretches of arginine- and lysine- rich sequences. These basic-residue stretches function as nuclear localization signals (NLSs) for translocation of the core protein to the nucleus (Chang et al., 1994; Suzuki et al., 1995). Each of the NLS motifs of the core protein is able to bind importin-α. At least two of them are required for efficient nuclear distribution of the core protein in cells, suggesting that they constitute a bipartite NLS (Suzuki et al., 2005).

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The major fate of core that is targeted to the nucleus is degradation by the nuclear proteasome (Hope et al., 2002; McLauchlan et al., 2002; Moriishi et al., 2003). Whether this is a cellular protein "quality control" mechanism, a normal pathway for core, or a pathway with other functional consequences is unclear. Nevertheless, it appears that constructs encoding mutations in the C terminus of core are less stable in cells than is wild-type core (Moriishi et al., 2003). McLauchlan and colleagues have proposed that the ability of domain II to mediate attachment of core to lipid droplets also protects core from degradation. Furthermore, they demonstrated that core constructs encoding a deletion in domain II are protected from degradation when they also encode a mutation that blocks cleavage of domain III by SPP (McLauchlan et al., 2002). Related to this observation, the mature form of core is much less stable when expressed as such than when expressed as the immature form of core which transiently contains domain III (E1 SS) before undergoing processing (Suzuki et al., 1995; Suzuki et al., 1999; Suzuki et al., 2001). Therefore, while the final product is the same, the presence of domain III during core biogenesis greatly influences core stability. Domain III, while not present in the mature wild-type core protein, plays a complex and important role in core stability. Like domain II, domain III and its cleavage may be involved in linking HCV core to cellular pathways that target it to other regions of the cell and protect it from degradation. Interestingly, although truncations and deletions in domain II lead to rapid degradation in mammalian cells, this phenomenon is not seen in cell-free capsid assembly systems, even when mammalian cell extracts are used (Klein et al., 2005; Klein et al., 2004). This is likely due to the absence of the nucleus in these systems, which prevents targeting to the nuclear proteasome, and allows such mutants to be expressed and analyzed.

Core appears to be peripherally associated with mitochondria, since it is accessible to protease digestion and carbonate extraction (Schwer et al., 2004) as is the case at the ER (McLauchlan et al., 2002). Most likely, core traffics from the ER to both the mitochondria and lipid droplets via membrane bridges, since both of these compartments are likely derived from the ER. Mitochondrial targeting appears to be governed by an aa sequence in core. Schwer et al. demonstrated that a short stretch extending from aa 149-158 located in domain II governs mitochondrial targeting (Schwer et al., 2004). Suzuki et al. reported that a region of 41 residues from aa 112-152 is responsible for association between the core protein and mitochondria (Suzuki et al., 2005). This discrepancy may be due to the differences of HCV clones and experimental settings.

POST-TRANSLATIONAL MODIFICATIONS OF HCV COREPost-translational modification plays crucial roles for regulating the function of the proteins. Several studies have shown post-translational modification of HCV core protein. Phosphorylation of the core protein in insects cells (Lanford et al., 1993),

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reticulocyte lysates (Shih et al., 1995), and mammalian cells (Lu and Ou, 2002) have been reported. Cellular protein kinase A (PKA) and protein kinase C (PKC) were identified as possible protein kinases responsible for phosphorylation of HCV core protein. Phosphorylation at Ser-116 may regulate nuclear localization of the core protein (Lu and Ou, 2002).

Post-translational modification of the core protein by tissue transglutaminase has been reported (Lu et al., 2001). Tissue transglutaminase catalyzes the formation of a γ-carboxyl-ε-lysine isopeptide bond by joining the γ-carboxamide group of glutamine to the amino group of lysine. A small fraction of the core protein has been shown to form a dimer that is highly stable and resistant to denaturation and reduction by SDS and β-mercaptoethanol. A potential role for tissue transglutaminase in core dimer formation has been proposed (Lu et al., 2001). The ubiquitin-proteasome pathway is the major route by which selective protein degradation occurs in eukaryotic cells and is now emerging as an essential mechanism of cellular regulation (Finley et al., 2004; Hershko and Ciechanover, 1998). As mentioned above, the core protein is targeted for ubiquitination and degradation by an unknown ubiquitin ligase. The C-terminus of the core protein is important for regulating stability of the protein (Kato et al., 2003; Suzuki et al., 2001). When the core protein is expressed as the C-terminal truncated forms such as aa 1-173 (21kDa) and 1-152 (17kDa), the core protein is unstable (Kato et al., 2003; Moriishi et al., 2003; Suzuki et al., 2001). Specific proteasome inhibitors stabilize these short-lived forms of the core protein, suggesting that the proteasome machinery is responsible for their degradation (Fig. 2). By contrast, the full-length form of the core protein (aa 1-191) is long-lived. Only the C-terminal truncated form of the core protein can be multi-ubiquitinated, and the predominant stable form of the core protein links to a single or only a few ubiquitin moieties (Suzuki et al., 2001). To understand the mechanism of ubiquitination of the core protein, the specific E3 ubiquitin ligase that acts on HCV core has to be identified.

A proteasome activator, PA28γ, has been identified as a core-binding protein by yeast two-hybrid screening (Moriishi et al., 2003). PA28γ can interact with the core protein in cultured cells, as well as in the liver of transgenic mice and chronic hepatitis C patients. PA28γ predominates in the nucleus and forms a homopolymer, which associates with the 20S proteasome (Tanahashi et al., 1997), thereby enhancing proteasome activity (Realini et al., 1997). Over-expression of PA28γ enhanced proteolysis of the core protein, suggesting that PA28γ affects proteasomal activity and regulates stability of the core protein (Moriishi et al., 2003) (Fig. 2). Evidence has been accumulating that ubiquitin-proteasome pathway plays a crucial role in the viral life cycle and in pathogenesis (Banks et al., 2003; Scheffner et al., 1993). However, the biological significance of ubiquitin-dependent degradation of the core protein remains to be elucidated.

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HCV CORE-HOST INTERACTIONSCore-host interactions will be discussed in terms of their affects on host antiviral and immune responses, and HCV pathogenesis. The recent finding of core protein in the serum on infected patients has forced one to think that HCV host interactions not only occur within infected cells, but they can also occur extracellularly.

EFFECTS ON T CELL FUNCTIONHCV infection in humans is almost invariably associated with viral persistence leading to chronic hepatitis – predisposing the host to development of cirrhosis

Fig. 2. A model for the processing of HCV precursor and degradation of the core protein by the Ubiquitin-proteasome pathway. The junction between core and E1 is cleaved by the signal peptidase, resulting in production of p23 form of the core protein. Additional cleavage of the core protein by signal peptide peptidase produces p21 form of the core protein. Further processed forms of the core protein, such as p17, are produced by unknown mechanisms. The C-terminal truncated form of the core protein is poly- ubiquitinated by an unidentified E3 ubiquitin ligase and targeted for proteasomal degradation. The immature core protein links to a single or a few ubiquitin moieties and is long-lived. A proteasome activator, PA28γ, enhances proteasomal degradation of the core protein.

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and hepatocellular carcinoma. CD8+ T cells play a pivotal role in controlling HCV infection; but, in chronic HCV patients, severe CD4+ and CD8+ T cell dysfunction has been observed (Shoukry et al., 2004). This suggests that HCV may employ mechanisms to evade or possibly suppress the host T cell response. In exploring the possible evasion mechanism(s) in order to design strategies for therapeutics and improved immunization, the HCV core protein was identified as an immunomodulatory molecule suppressing T lymphocyte responsiveness through its interaction with complement receptor (gC1qR) (Kittlesen et al., 2000).

It was demonstrated that the HCV core protein suppresses an in vivo anti-viral CD8+ T cell response to vaccinia virus, and inhibits the production of IFN-γ and IL-2 in an experimental murine model. A host target protein (gC1qR) on T cells was shown to bind HCV core. Like the natural ligand, C1q, the binding of extracellular core to gC1qR displayed on T cell surface lead to CD4+ T cell deregulation and suppression of CD8+ T cell function. Importantly, HCV core-gC1qR ligation induced the expression of negative signaling molecules (e.g. SHP-1 and SOCS1) in CD4+ T cells. The data suggest that core has potent immunomodulatory functions.

EFFECTS ON TOLL-LIKE RECEPTORSCells sense the presence of extracellular pathogens via cell surface toll-like receptors (TLRs). There are approximately 10-15 TLRs in mammals, which are responsible for sensing microbial infection, via recognition of pathogen associated molecular patterns (PAMP), such as lipopolysaccharide (LPS; TLR4), double-stranded RNA (dsRNA; TLR3), CpG DNA of bacteria (TLR9), and single-stranded RNA (ssRNA; TLR7) (Iwasaki and Medzhitov, 2004). After binding pathogens, TLR signaling involves coupling of toll-IL-1 receptor (TIR) containing adapter proteins such as TIRAP, TRIF, TIRAP and MAL, and activation of signaling molecules IL-1 receptor associated kinase (IRAK), MyD88, and TNF receptor-associated factor 6 (TRAF-6). Ultimately, transcription factors such as mitogen activated protein kinases (MAPK), NF-κB, and IRF-3 become activated, leading to production of IFN-α/β (Hertzog et al., 2003). Interestingly, DC maturation in vitro is impaired in chronic HCV infection when compared to those subjects with spontaneously resolved infection and normal controls (Anthony et al., 2004; Dolganiuc et al., 2003; Kanto et al., 2004; Murakami et al., 2004a; Sarobe et al., 2003; Tsubouchi et al., 2004a; Tsubouchi et al., 2004b; Wertheimer et al., 2004). Recent studies have provided mechanistic insights into these events.

In a study of the effect on the immunostimulatory effects of lipopeptides, 10 of 14 and 9 of 14 HCV core lipopeptides stimulated a reporter gene in TLR2-expressing and TLR4-expressing cells but not in mock-transfected control cells (Duesberg et al., 2002). However, activation was dependent on the lipid moiety since the same free peptides had no stimulatory effect on the TLR2 or TLR4 transfected

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cells. A study by a different group found that addition of recombinant HCV core protein to human monocytes, and human embryonic kidney cells transfected with TLR2 triggered inflammatory cell activation and failed to activate macrophages from TLR2 or MyD88-deficient mice (Dolganiuc et al., 2004). HCV core induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Cell activation required core aa 2-122. Interestingly, HCV core protein was also taken up by macrophages, but this was independent of TLR2 expression. These data indicate that the HCV core protein can trigger innate immune responses.

EFFECTS OF HCV CORE ON THE INTERFERON SYSTEMSeveral studies have documented that the HCV core protein can activate the interferon (IFN) system. For example, core activates the IFN stimulated genes (ISG) 2-5 OAS (Naganuma et al., 2000) and PKR (Delhem et al., 2001). PKR and 2-5 OAS are two major ISGs that mediate the IFN antiviral response against many viruses. It was also recently shown that HCV core protein activates the innate antiviral cellular response involving interferon regulatory factors (Miller et al., 2004). Core induced IRF-1 transcription and mRNA expression, and caused dose-dependent induction of the IFN-β promoter and IFN-β mRNA expression. In the presence of IFN-α, core expression caused increased IFN-stimulated gene factor 3 (ISGF3) binding to the IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1. Core expression also activated IFN-γ signaling (Miller et al., 2004).

The effects of core on innate cellular antiviral responses including TLR and IFN pathways may be critically important during acute infection. Following binding, internalization, and uncoating of HCV virions, core, in the form of nucleocapsid, is the first viral protein to interact with the intercellular milieu of cellular proteins and signaling pathways. Because core mutates during virus replication, HCV core is present as a quasispecies in infected patients (Pawlotsky, 2003). What is not clear at present is whether HCV core's inherent variability influences innate antiviral responses such as TLR signaling and IRF-Jak-Stat activation. Fig. 3 suggests that there is indeed heterogeneity in innate antiviral responses to genetically different HCV core isolates. Fig. 3A depicts the sequence of 2 core proteins (named Core 1 and Core 2) derived from 2 different genotype 1b infected patients. As shown in the figure, the two isolates differed by 7 aa. The 2 core genes were engineered into a tetracycline regulated expression vector, such that in the absence of tetracycline in the medium, both Core 1 and Core 2 proteins were expressed in HeLa cells. Addition of tetracycline to the medium blocked core expression. Fig. 3C presents the effects of Core 1 and Core 2 expression on transcription of an IFN responsive promoter, the ISRE. In the absence of IFN, expression of Core 1 was associated with a 3-fold increase in activation of the ISRE, compared to when

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gene expression was repressed. In the presence of IFN, Core 1 induced a 2-fold increase in luciferase activity. Expression of Core 2 resulted in only marginal ISRE stimulation. These data demonstrate that 2 genetically different HCV core proteins activate a canonical IFN promoter to varying degrees. The data suggest that HCV quasispecies differentially modulate host cell responses. Indeed, other studies have demonstrated that NS5A mediated transcriptional activation varies among clinical quasispecies isolates (Pellerin et al., 2004). Thus, future studies should take into account genetic and structural heterogeneity of HCV isolates as being important factors in host responsiveness to HCV infection.

This concept may have clinical implications. It can be hypothesized that genetic and structural variants of HCV proteins such core could differentially trigger

Fig. 3. Effects of HCV Core Protein Expression on Type I IFN Signal Transduction. A, the sequence of the Core 1 and Core 2 genes are aligned. B, Tetracycline regulated expression of the Core 1 and Core 2 proteins in HeLa cells. Plasmids were transfected into HeLa tet-off cells, grown in the absence and presence of tetracycline to induce and repress core protein expression, respectively, and protein lysates were subjected to Western blot analysis at 48 hours post-transfection. C, Differential effects of Core 1 and Core 2 proteins on ISRE activation. pTRE-Core 1 and pTRE-Core 2 plasmids were cotransfected with an ISRE-luciferase reporter plasmid into HeLa tet-off cells, incubated in the presence or absence of tetracycline for 40 hours, and treated with or without 500 U/ml of IFN-α for 6 hours. Luciferase activity was determined on equal amounts of protein lysates.

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innate antiviral responses during acute infection. Thus, some HCV infections may be "silent" because they minimally activate the TLR and/or IFN cellular defense systems. This would have obvious selective advantage for the virus and could contribute to the establishment of chronic infection. Alternatively, when a virus enters cells in a "noisy" fashion, it has a poor chance of establishing chronic infection because the innate antiviral responses would quickly shut down virus replication. Finally, stimulation of the IFN system by the HCV core protein may be required to balance the anti-IFN functions of other HCV proteins such as E2 (Taylor et al., 1999), NS5A (Gale et al., 1997; Polyak et al., 2001), and NS3 (Foy et al., 2003) during certain stages of the HCV replication cycle.

EXTRACELLULAR VERSUS INTRACELLULAR EFFECTS OF COREHCV core is found within infected cells as well as in patient serum (Kashiwakuma et al., 1996; Widell et al., 2002). Extracellular core protein likely affects the modulation of T cell function, TLR signaling and DC function as described above. Thus, it is important to consider the contribution of extracellular and intracellular core protein to the biological activity in question. Indeed, CD81 engagement by the HCV envelope glycoprotein E2 inhibits NK and T cell cytotoxic function and signal transduction (Crotta et al., 2002; Tseng and Klimpel, 2002; Wack et al., 2001), and induced pro-inflammatory chemokine expression in hepatocytes (Balasubramanian et al., 2003). Thus, immune function may be altered as cells "sample" the microenvironment through HCV-host interactions that are limited to molecules on the cell surface, such as the HCV core-TLR or core-C1qR interaction. Moreover, these extracellular HCV-host interactions may also contribute to HCV pathogenesis.

HCV CORE AND PATHOGENESISRecent work has demonstrated that the HCV core protein may also participate in the pathogenesis of liver disease. Development of fibrosis is characterized histologically with infiltration of inflammatory lymphocytes, hepatocellular apoptosis, and Kupffer cell activation. HSC proliferate and undergo and become highly activated, which involves secretion of large amounts of extracellular matrix proteins (Bataller and Brenner, 2005). Despite a large body of literature from clinical and animal studies on fibrosis development, very little is known about how HCV causes fibrosis.

A recent study found that addition of recombinant core protein to activated human hepatic stellate cells (HSC) stimulated intracellular signaling pathways, while viral transduction of HCV core into HSCs caused increased cell proliferation (Bataller et al., 2004). Interestingly, the HSC response appeared to differ between core and other HCV non-structural proteins. The data suggest that HCV core and non-structural proteins can modulate the activity of HSC, which may contribute to fibrosis. This study also reinforces the notion that HCV proteins can have intracellular as well as extracellular effects on a variety of cells.

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A second important point from the study of Bataller et al., (Bataller and Brenner, 2005), is that HCV proteins including core induce oxidative stress on HSC which is involved in HSC activation. Indeed, antioxidant therapy reduces the effects of HCV proteins on HSCs. This finding is in line with the current thinking that oxidative stress is central to induction of fibrosis in many model systems. HCV core induced oxidative stress also affects mitochondrial physiology.

HCV CORE AND MITOCHONDRIAL DYSFUNCTIONExpression of HCV core protein in transgenic mice and in cell culture induces oxidative stress. It has been shown that core protein localizes to mitochondria, between the mitochondrial outer membrane and ER (Moriya et al., 1998; Moriya et al., 2001a; Okuda et al., 2002; Schwer et al., 2004; Suzuki et al., 2005), as described in the targeting section above. Core protein expression and mitochondrial localization inhibits electron transport at complex I, increases complex I reactive oxygen species (ROS) production, decreases mitochondrial glutathione, and increases mitochondrial permeability transition in response to exogenous oxidants such as alcohol (Korenaga et al., 2005; Okuda et al., 2002; Wen et al., 2004). These effects are associated with increased hepatocyte apoptosis in the presence of HCV core protein, ethanol and cytochrome P4502E1. Like the case with HSC, core and ethanol metabolism effects on apoptosis can be prevented with antioxidants (Otani et al., 2005).

HEPATITC STEATOSIS AND HEPATOCARCINOGENESISEvidence has been accumulating that HCV core protein is directly involved in pathogenesis (Giannini and Brechot, 2003; McLauchlan, 2000). As shown in Table 1, many cellular proteins, which interact with core protein have been identified. Several studies have suggested that the core protein plays a crucial role for hepatocarcinogenesis (Chang et al., 1998; Moriya et al., 1998; Ray et al., 1996).

Recent studies have highlighted steatosis as a basis of HCV-associated HCC (Lerat et al., 2002; Moriya et al., 1998). Steatosis, which is an accumulation of fat deposits in hepatocytes, is one of the histological features of chronic hepatitis C (Bach et al., 1992; Lefkowitch, 2003). In vitro studies have shown that HCV core protein associates with cellular lipid droplets, via direct interaction with apolipoprotein A2 (Barba et al., 1997; Shi et al., 2002). The mice transgenic for HCV core gene have been shown to develop steatosis and hepatocellular carcinoma (HCC) (Moriya et al., 1998; Moriya et al., 1997b). Steatosis in the core-transgenic mice is age-dependent and characterized by the appearance of micro- and macro-vesicular lipid droplets (Moriya et al., 1998). Lerat et al. have confirmed that transgenic mice expressing the whole genome of HCV also develops steatosis and HCC (Lerat et al., 2002).

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Although the molecular mechanisms of steatosis caused by the core protein is still unclear, the core protein may alter lipid metabolism by interacting with cellular proteins involved in lipid accumulation and storage in hepatocytes (Barba et al., 1997; Sabile et al., 1999; Shi et al., 2002). The concentration of carbon 18 monosaturated fatty acids were increased in the livers of the core-transgenic mice and chronic hepatitis C patients, suggesting that HCV core affects a specific pathway in lipid metabolism (Moriya et al., 2001b). Nonetheless, transgenic mouse lines established by other groups did not show either steatosis nor HCC (Kawamura et al., 1997; Pasquinelli et al., 1997). These discrepancies suggest that not only the viral proteins but also other factors are involved in hepatocarcinogenesis. These discrepancies may be due to differences in genetic backgrounds of the mice and expression levels of the viral proteins.

Table 1. Cellular proteins that bind to the HCV core protein. The list contains cellular proteins with various cellular functions that interact with HCV core. The interaction of HCV core with these cellular proteins may have pathogenic implications. Please refer to the text for details.

Core-Interacting protein Function Reference

Apolipoprotein AII lipid metabolism Sabile et al., 1999; Shi et al., 2002

CAP-Rf RNA helicase You et al., 1999

complement receptor gC1qR T-cell response Kittlesen et al., 2000

cyclin-dependent kinase 7 cell cycle Ohkawa et al., 2004

DEAD box protein RNA helicase Mamiya and Worman, 1999

DEAD box protein 3 RNA helicase Owsianka and Patel, 1999heterogeneous nuclear ribonucleoprotein K transcriptional control Hsieh et al., 1998

JAK1/2 signal transduction Hosui et al., 2003

lymphotoxin-β receptor cytotoxicity Chen et al., 1997

p53 transcriptional control Otsuka et al., 2000

p73 transcriptional control Alisi et al., 2003

proteasome activator PA28γ protein stability Moriishi et al., 2003

retinoid X receptor α transcriptional control Tsutsumi et al., 2002

Smad3 transcriptional control Cheng et al., 2004

Sp110b transcriptional control Watashi et al., 2003

STAT3 cell transformation Yoshida et al., 2002

TAFII28 transcriptional control Otsuka et al., 2000

Tumor necrosis factor receptor 1 apoptosis Zhu et al., 2001

14-3-3 protein signal transduction Aoki et al., 2000

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ACKNOWLEDGEMENTSSJP is partially supported by NIH grants AA13301 and DK62187, and the University of Washington Royalty Research Fund. JRL received support from Puget Sound Partners, and KCK received support from NIH training grant T32 CA09229. IS and TM are supported in part by grants from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, RandD Promotion and Product Review of Japan (ID:01-3) and the Second Term Comprehensive 10-year Strategy for Cancer Control of the Ministry of Health, Labor, and Welfare of Japan. IS and TM also thank their colleagues, T.Tsutsumi, K.Ishii, H.Aizaki, K. Murakami, R.Suzuki, T, Suzuki, (National Institute of Infectious Diseases), Y.Shintani, H.Fujie, K. Moriya, K.Koike, (Tokyo University) and K.Moriishi, Y. Matsuura (Osaka University) for contribution.

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HCV Envelope Glycoproteins

Chapter 4

HCV Glycoproteins: Assembly of a Functional E1-E2 Heterodimer

Muriel Lavie, Anne Goffard and Jean Dubuisson

ABSTRACTThe two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1-E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1-E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have been limited by the lack of a robust cell culture system for HCV replication and viral particle production. Recently, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins, allowing the characterization of functional E1-E2 envelope glycoproteins. Here, we review our understanding to date on the assembly of the functional HCV glycoprotein heterodimer.

INTRODUCTIONAs obligate intracellular parasites, all viruses have evolved ways of entering target cells to initiate replication and infection. The first step in virus entry is the recognition of host cells through cell surface receptor(s). This initial engagement can mediate attachment as well as act as a primer for subsequent conformational alteration, leading to virus entry into host cell. In many cases, interaction with a receptor is important for defining the tropism of a virus for a particular organism, tissue or cell type. Enveloped viruses possess a lipid bilayer that surrounds their nucleocapsid. The glycoproteins present in their envelope are involved in the receptor-binding step. After attachment, the entry of these viruses into cells requires the fusion of the viral and a cellular membrane by a process that is also driven by the viral envelope glycoproteins. To fulfill these functions, viral envelope glycoproteins have to adopt dramatically different conformations during the virus lifecycle. In addition, these conformational changes have to occur at a precise time of the virus lifecycle, and thus, have to be tightly modulated.

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HCV encodes two envelope glycoproteins, named E1 and E2. For a long time, the lack of a cell culture system supporting efficient HCV replication and particle assembly has hampered the characterization of the envelope proteins present on the virion. Cell culture transient expression systems have allowed investigators to characterize the first steps in the biogenesis of HCV envelope glycoproteins (reviewed in: Op De Beeck et al., 2001). In addition, surrogate models have also been developed to study the entry steps of HCV lifecycle (reviewed in: Op De Beeck and Dubuisson, 2003). However, it is only recently that a model mimicking the entry process of HCV lifecycle has been developed. This has been achieved by pseudotyping retroviral particles with native HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). This new tool allows, for the first time, the characterization of the assembly of functional HCV envelope glycoproteins.

BIOGENESIS OF HCV ENVELOPE GLYCOPROTEINS

CLEAVAGE OF HCV GLYCOPROTEINS FROM THE VIRAL POLYPROTEINAs for the other members of the Flaviviridae family, the genome of HCV encodes a single polyprotein. This ~3010 amino acid polyprotein is processed by cellular (signal peptidase and signal peptide peptidase) and viral proteases (NS2-3 and NS3-4A) to generate at least 10 polypeptides (reviewed in: Penin et al., 2004). The nonstructural proteins are released from the polyprotein after cleavage by HCV proteases NS2-3 and NS3-4A, whereas the structural proteins are released by host endoplasmic reticulum (ER) signal peptidase(s) (Fig. 1)(reviewed in Reed and Rice, 2000). Further processing mediated by a signal peptide peptidase also occurs at the C-terminus of the capsid protein (McLauchlan et al., 2002). Most cleavages in the polyprotein precursor proceed to completion during or immediately after translation (Grakoui et al., 1993; Dubuisson et al., 1994; Lin et al., 1994; Mizushima et al., 1994; Dubuisson and Rice, 1996). Partial cleavages occur at the E2/p7 and p7/NS2 sites, leading to the production of an uncleaved E2p7NS2 molecule. While most of NS2 is progressively cleaved from the E2p7NS2 precursor, the cleavage between E2 and p7 does not change over time, at least for most HCV strains analyzed (Dubuisson, 2000). Thus, this results in cleavage products consisting of E2, E2p7, p7, and NS2.

The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can efficiently function as signal peptides. Indeed, when fused to a reporter protein, the signal peptides of p7 and NS2 are efficiently cleaved (Carrère-Kremer et al., 2004). These data indicate that inefficiency of cleavage at E2/p7 and p7/NS2 sites is not due to the presence of suboptimal signal peptides. The p7 polypeptide is a polytopic membrane protein containing two transmembrane domains with both its N- and C-termini oriented toward the ER lumen (Fig. 1)(Carrère-Kremer et al.,

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2002). Interestingly, the presence of the fi rst transmembrane domain of p7 reduces the effi ciency of p7/NS2 cleavage (Carrère-Kremer et al., 2004). Sequence analyses and mutagenesis studies have also identifi ed structural determinants responsible for the partial cleavage at both E2/p7 and p7/NS2 sites (Carrère-Kremer et al., 2004). In addition, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane α-helix located downstream of the cleavage sites might impose additional structural constraints to these cleavage sites (Fig. 1). Such constraints in the processing of a polyprotein precursor are likely essential for HCV to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

The processing at the E2p7 site has been further explored. It has been reported to be more effi cient in genotype 1b (strain BK) than in the genotype 1a (strain H77c) (Dubuisson et al., 1994; Lin et al., 1994). A sequence comparison of p7 signal peptides of these two viral strains has identifi ed a difference of 3 amino acids and mutational analysis has shown that the V720L change in the H77c sequence substantially increases the effi ciency of processing at the E2/p7 site (Isherwood and Patel, 2005). Although, when expressed alone, p7 protein has been shown to adopt a double membrane spanning topology with both extremities orientated luminally in the ER (Carrère-Kremer et al., 2002), the C-terminal part of E2p7 proteins has been found to be located in the cytosol (Isherwood and Patel, 2005). These data suggest

Fig. 1. Processing of the N-terminal one-third of HCV polyprotein. The arrows show host signal peptidase cleavages. Partial cleavages at E2/p7 and p7/NS2 sites are indicated by dotted arrows. Cleavage by the host cell signal peptide peptidase (SPP) is indicated by scissors. The signal peptide and signals of reinitiation of translocation are shown as a black cylinder and light grey cylinders, respectively. The transmembrane domains of HCV envelope glycoproteins are represented in their pre-cleavage topology. Post-cleavage reorientation of the glycoprotein signals of reinitiation of translocation is indicated by curved arrows.

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that p7 can potentially adopt a dual transmembrane topology. It remains, however, to be shown whether an E2p7 with a cytosolic orientation of the C-terminus of p7 exists when this protein is expressed in the context of the polyprotein.

Since p7 and NS2 are not essential for HCV genomic replication (Lohmann et al., 1999; Blight et al., 2000), they will likely play their role in virion assembly, a process that is supposed to be tightly regulated. It has recently been shown that p7 reconstituted into artificial lipid membranes homo-oligomerizes and behaves as an ion channel protein (Griffin et al., 2003; Pavlovic et al., 2003; Premkumar et al., 2004). It is likely that, when bound to E2, p7 cannot oligomerize and function as an ion channel, and the existence of E2p7 would therefore reduce the amount of functional p7 molecules available. Production of precursors like E2p7NS2 and E2p7 might be a means to maintain p7 inactive during the phase of the accumulation of E2 molecules required for HCV envelope formation. Alternatively, such precursors might also control the temporal release of E2 and NS2.

GLYCOSYLATION OF HCV ENVELOPE GLYCOPROTEINSN-linked glycosylation is one of the most common types of protein modification, and it occurs by the transfer of an oligosaccharide from a lipid intermediate to an Asn residue in the consensus sequence Asn-X-Thr/Ser of a nascent protein, where X is any amino acid except Pro (Kornfeld and Kornfeld, 1985; Gavel and von Heijne, 1990). The addition of this glycan is catalyzed by the oligosaccharyltransferase, which is closely associated with the translocon through which the nascent peptidic chains emerge in the ER lumen (Silberstein and Gilmore, 1996). However, not every tripeptide sequence in a protein sequence is used for carbohydrate addition (Gavel and von Heijne, 1990). In the early secretory pathway, the glycans play a role in protein folding, quality control and certain sorting events. Viral envelope proteins usually contain N-linked glycans that can play a major role in their folding, in their entry functions or in modulating the immune response (Hebert et al., 1997; Ohuchi et al., 1997a; Ohuchi et al., 1997b; van Kooyk and Geijtenbeek, 2003; von Messling and Cattaneo, 2003; Wei et al., 2003).

The ectodomains of HCV envelope glycoproteins E1 and E2 are highly modified by N-linked glycans. E1 and E2 possess up to 6 and 11 potential glycosylation sites, respectively (Fig. 2). Sequence analyses of E1 indicate that 5 potential N-glycosylation sites are strongly conserved among HCV genotypes (Goffard and Dubuisson, 2003; Zhang et al., 2004b). However, in one case the presence of a proline residue immediately downstream the glycosylation site is unfavorable for glycosylation, and it has been confirmed experimentally that this site is not glycosylated (Meunier et al., 1999). Interestingly, the glycosylation site of E1 at position 250 is poorly conserved; this site is indeed only observed in genotypes 1b and 6 (Fig. 2)(Goffard and Dubuisson, 2003). Most E2 glycosylation sites are

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also well conserved. Indeed, global sequence analyses of potential glycosylation sites in E2 indicate that nine of the eleven sites are strongly conserved (Goffard and Dubuisson, 2003; Zhang et al., 2004b). The two remaining sites, N5 and N7, show conservation levels of 75% and 89%, respectively (Goffard and Dubuisson, 2003).

Mutants of E1 and E2 have been produced to characterize the glycosylation of these proteins (Meunier et al., 1999; Nakano et al., 1999; Slater-Handshy et al., 2004; Goffard et al., 2005). In the context of the H strain, the 4 potential glycosylation sites of E1 were shown to be occupied by glycans (Meunier et al., 1999; Goffard et al., 2005). In the case of E2, a fi rst study has shown that mutation of some glycosylation sites in the context of a truncated form of E2 alters the recognition by sera from HCV patients (Nakano et al., 1999); however, these mutants were not characterized in terms of glycosylation and no clear conclusion can be drawn from this study. More recently, glycosylation mutants have been produced in the context of a truncated form of E2 ending at position 660 (Slater-Handshy et al., 2004). The E2 sequence of HCV isolate used in this study contains 10 instead of 11 potential

Fig. 2. Schematic representation of E1 and E2 features. Positions of N-linked glycans are indicated as an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein. The numbers correspond to the positions in the polyprotein of reference strain H (acc. Number AF009606). Glycans involved in HCVpp entry are indicated with a black square (Goffard et al., 2005). Glycosylation sites for which the mutation alters E1E2 folding are indicated with a grey circle (Goffard et al., 2005). The hypervariable region 1 (HVR1) of E2 is shown as a grey box. The black boxes correspond to E2 epitopes recognized by neutralizing antibodies (Hsu et al., 2003). The sequences of the transmembrane domains of HCV envelope glycoproteins are indicated above their corresponding region in E1 and E2. The two hydrophobic segments in these regions are underlined. The charged residues present between the two hydrophobic stretches are in white lettering. Arrows indicate the positions of inserted alanine residues that disrupt HCV E1E2 heterodimerization (Op De Beeck et al., 2000).

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glycosylation sites, the site N5 at position 476 being missing (Fig. 2). Interestingly, the last two glycosylation sites, N10 and N11, were not occupied in E2660 (Slater-Handshy et al., 2004). However, at least one of these sites was occupied in the context of full-length E2. A more recent mutagenesis study, in the context of an E2 glycoprotein containing 11 potential glycosylation sites, has shown that all the sites are occupied by glycans (Goffard et al., 2005). In this case, E2 was expressed as a polyprotein containing full-length E1 and E2. Altogether, these data indicate that full-length and truncated forms of E2 can have different properties.

The addition of the glycan precursor is catalyzed by the oligosaccharyltransferase, and this enzyme is thought to have access only to nascent chains as they emerge from the ribosome at the luminal face of the rough ER (Silberstein and Gilmore, 1996). The glycosylation process of HCV envelope glycoprotein E1 has been analyzed in the context of a Man-P-Dol-deficient cell line (B3F7) and it has been shown to occur post-translationally (Duvet et al., 2002), indicating that the oligosaccharyltransferase has also access to the E1 glycoprotein for more than an hour after its translation. A characterization of HCV glycoprotein E1 has also shown that, in the absence of E2, different glycoforms of E1 are produced and the glycosylation of E1 is improved by co-expression of E2 in cis (Dubuisson et al., 2000).

FOLDING OF HCV ENVELOPE GLYCOPROTEINSHCV envelope glycoproteins have been shown to assemble as a noncovalent E1E2 heterodimer (Deleersnyder et al., 1997). However, at least in heterologous expression systems, HCV envelope glycoproteins have a tendency to also form misfolded aggregates stabilized by disulfide bonds (reviewed in (Dubuisson, 2000)). Analyses of HCV envelope glycoproteins with conformation-sensitive antibodies are therefore necessary to discriminate noncovalent heterodimers from misfolded complexes (Deleersnyder et al., 1997; Cocquerel et al., 2003b). Alternatively, such discrimination can also be made by analyzing disulfide-bond formation by migrating HCV envelope glycoproteins on SDS-PAGE under non-reducing conditions (Dubuisson and Rice, 1996; Brazzoli et al., 2005). Analyses of the formation of conformation-dependent epitopes and disulfide-bond formation indicate that folding of HCV envelope glycoproteins is a slow process (Deleersnyder et al., 1997; Dubuisson and Rice, 1996; Duvet et al., 1998; Brazzoli et al., 2005). Interestingly, the folding of E1 has been shown to be dependent on the co-expression of E2 (Michalak et al., 1997; Patel et al., 2001). In addition, it has also been shown that the folding of E2 is also dependent on the co-expression of E1 (Cocquerel et al., 2003a; Brazzoli et al., 2005). Altogether, these observations indicate that HCV envelope glycoproteins cooperate for the formation of a functional complex. These observations also indicate that, although some degree of folding can be observed in E2 expressed alone (Michalak et al., 1997; Cocquerel et al., 2003a), both glycoproteins need to be co-expressed to analyze their functional properties.

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During their folding, HCV envelope glycoproteins have been shown to interact with calnexin (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001; Brazzoli et al., 2005), a lectin-like ER chaperone, which shows an affinity for monoglucosylated N-linked oligosaccharides (Trombetta and Helenius, 1998). Both E1 and E2 have been found to associate rapidly with calnexin and dissociate slowly, suggesting a role of this chaperone in the folding of HCV envelope glycoproteins (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001). However, more recent data suggest that only E1 interacts with calnexin (Brazzoli et al., 2005). Differences in the cell lines used and/or in the levels of expression of the envelope glycoproteins might potentially explain these discrepancies. Further experiments in cell cultures infected with native HCV particles will be needed to confirm the involvement of calnexin in the folding E2.

The presence of glycans on HCV envelope glycoproteins can potentially affect their folding either directly or through interaction with calnexin. Site-directed mutagenesis studies have indeed shown that the absence of some glycans in E1 (N1 and N4) and E2 (N8 and N10) leads to misfolding of HCV envelope glycoproteins (Fig. 2)(Meunier et al., 1999; Goffard et al., 2005). This alteration in folding was not due to the lack of interaction of HCV envelope glycoproteins with calnexin, suggesting that the mutations would rather have a direct effect on protein folding. The presence of a large polar saccharide is indeed known to affect the folding at least locally by orienting polypeptide segments toward the surface of protein domains (Imperiali and O'Connor, 1999; Wormald and Dwek, 1999).

INVOLVEMENT OF THE TRANSMEMBRANE DOMAINS IN THE BIOGENESIS OF E1E2 HETERODIMER

MEMBRANE ANCHOR AND SIGNAL SEQUENCE

Due to their resistance to alkaline or salt extraction, HCV envelope glycoproteins have been confirmed to be membrane associated proteins (Ralston et al., 1993; Cocquerel et al., 2001). In addition, deletion of the C-terminal hydrophobic regions of these proteins leads to their secretion, indicating that these regions are involved in membrane anchoring (Michalak et al., 1997). Sequence analysis of a large number of HCV isolates has shown that the C-termini of E1 and E2 contain hydrophobic sequences that are less than 30 amino acid residues long (Fig. 2)(Cocquerel et al., 2000). As in other viruses of the Flaviviridae family, these regions are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved positively charged residue (Cocquerel et al., 2000). Interestingly, when fused to a reporter protein the second hydrophobic stretch functions as a signal sequence (Cocquerel et al., 2002), which is in agreement with the observation that HCV envelope glycoproteins are released from the polyprotein precursor after cleavage by host signal peptidase(s) (Dubuisson et al., 2002). It is worth noting that in the context of HCV polyprotein, only the sequence located at

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the C-terminus of the immature form of the capsid protein is a true signal peptide that will interact with the signal recognition particle (Santolini et al., 1994). The sequence present at the C-terminus of E1 and E2 do not interact with the signal recognition particle, and they should be called signals of reinitiation of translocation (Fig. 1). Deletion of these signals leads to the secretion of E1 and E2, indicating that these signals are involved in their membrane anchoring (Cocquerel et al., 2000).

ER RETENTION FUNCTION

HCV envelope glycoproteins are retained in the ER (Dubuisson et al., 1994; Deleersnyder et al., 1997; Duvet et al., 1998), and ER retention signals are present in the transmembrane domains of E1 and E2 (Cocquerel et al., 1998; Cocquerel et al., 1999). In addition, the charged residues of the transmembrane domains of E1 (Lys) and E2 (Asp and Arg) play a key role in the ER retention of these glycoproteins (Cocquerel et al., 2000). It has been proposed that an additional ER retention signal might also be present in the ectodomain of E1 (Mottola et al., 2000). Interestingly, in some conditions of overexpression a small fraction of HCV envelope glycoproteins has been shown to accumulate at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). Cell surface expression of E1 and E2 is likely due to the accumulation of small amounts of glycoproteins escaping the ER-retention machinery, due to saturation of this mechanism.

ROLE IN HETERODIMERIZATION

In addition to their anchoring, signal sequence and ER retention functions, the transmembrane domains of HCV envelope glycoproteins have also been shown to play a major role in the assembly of E1E2 heterodimer. Indeed, deletion of the transmembrane domain of E2 or its replacement by the anchor signal of another protein abolishes the formation of E1E2 heterodimer (Selby et al., 1994; Michalak et al., 1997; Cocquerel et al., 1998; Patel et al., 2001). Other studies by site-directed mutagenesis or alanine scanning insertion mutagenesis (Cocquerel et al., 2000; Op De Beeck et al., 2000) have confirmed that the transmembrane domains of E1 and E2 play a direct role in E1E2 assembly. In addition, alanine scanning insertion mutagenesis allowed to identify two distinct segments in the transmembrane domain of E1 and one in the transmembrane domain of E2 that were specifically involved in E1E2 assembly (Fig. 2). Interestingly, at least one region located outside of the transmembrane domains has also been shown to be involved in heterodimerization (Drummer and Poumbourios, 2004).

TOPOLOGICAL CHANGE IN THE TRANSMEMBRANE DOMAIN OF HCV GLYCOPROTEINS

The topology of the transmembrane domain of HCV envelope glycoproteins has given rise to some controversy. Indeed the presence of a first hydrophobic stretch and a signal sequence function separated by charged residues in the transmembrane domains of E1 and E2 has suggested that they might be composed of two membrane

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spanning segments with the charged residues facing the cytosol (Charloteaux et al., 2002). This type of organization has been observed in the C-terminal region of the envelope glycoprotein E2 of the alphaviruses as well as for the envelope proteins of the flaviviruses (Strauss and Strauss, 1994; Op De Beeck et al., 2003; Zhang et al., 2003). However sequence analysis and data of alanine scanning insertion mutagenesis were in favor of a single spanning topology of E1 and E2 transmembrane domain (Cocquerel et al., 2000; Op De Beeck et al., 2000). A study of the topology of the transmembrane domains of HCV envelope proteins has been performed by determining the accessibility of their N- and C-termini in selectively permeabilized cells (Cocquerel et al., 2002). This work has shown that before signal sequence cleavage at their C-terminus, the transmembrane domains form a hairpin structure (Fig. 1). However, after cleavage between E1 and E2 or between E2 and p7, the second C-terminal hydrophobic stretch is reoriented towards the cytosol, leading to the formation of a single membrane-spanning domain. Here again, the charged residues located in the middle of the transmembrane domains were shown to play a crucial role in their structural dynamics (Cocquerel et al., 2002).

ROLE OF HCV ENVELOPE GLYCOPROTEINS IN VIRUS ENTRYFor most viruses, entry into the cytosol is a multistep process, during which the host cell assists the incoming virus. Viruses first attach themselves to components of the plasma membrane, which they use as non-specific attachment factors or as specific cell surface receptors. Viral attachment is mediated by the binding of a protein present at the surface of the virion to a molecule on the cell surface acting as a virus receptor. The envelope glycoprotein complex E1E2 is the viral component thought to be present at the surface of HCV particles and it is therefore the obvious candidate ligand for cellular receptors. Receptor binding can activate cellular endocytic pathways through which viruses are internalized in endosomes. When they reach the appropriate intracellular location, viruses are activated for penetration by cellular signals and make their way through the membrane of the endosome, or through the plasma membrane for those that do not enter by endocytosis. Enveloped viruses fuse their lipid envelope with the plasma membrane or the membrane of an endosome, resulting in the release of the nucleocapsid into the cytosol.

MODELS TO STUDY HCV ENTRYIn the absence of a robust cell culture system to amplify HCV, several models have been developed to study HCV entry. In a first approach, a soluble form of HCV glycoprotein E2 has been used to identify cell surface proteins potentially involved in HCV entry (Rosa et al., 1996; Pileri et al., 1998). Although this approach is potentially interesting in protein-protein interactions studies, it cannot be used to study the entire entry process. In addition, as discussed above, due to their cooperative role in folding, both glycoproteins need to be co-expressed to analyze their functional properties. To study the role of E1E2 envelope glycoproteins in HCV

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entry, several surrogate models of HCV particles have therefore been developed. As a first approach, virus-like particles have been produced in insect cells infected by a recombinant baculovirus containing the cDNA of HCV structural proteins (Baumert et al., 1998). However these particles are not infectious and they are retained in an intracellular compartment. It is therefore difficult to evaluate how close these virus-like particles are to native virion. In addition, due to the absence of infectivity, these particles cannot be used to study the fusion process. Another approach to study HCV entry has been to produce virosomes by incorporating E1E2 heterodimers into liposomes (Lambot et al., 2002). These virosomes can be used to study the interactions between E1E2 heterodimers and cell surface receptors. However, it has not been shown whether the envelope glycoproteins incorporated into these liposomes can induce fusion.

Other models have been based on pseudotyping of viral vectors. The first model that has been developed was based on vesicular stomatitis virus (VSV) pseudotyped with modified E1 and/or E2 glycoproteins (Lagging et al., 1998; Matsuura et al., 2001). In these particles, the transmembrane domains of HCV envelope glycoproteins have been replaced by the transmembrane domain and cytoplasmic tail of the VSV envelope glycoprotein G. This allows the export of HCV envelope glycoproteins to the cell surface (Takikawa et al., 2000). However, some doubts have been raised on the infectivity of such VSV pseudotyped particles (Buonocore et al., 2002). In addition, replacement of HCV envelope glycoproteins has been shown to alter their entry function (Hsu et al., 2003).

More recently, retroviruses have also been used to produce pseudotyped particles containing HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Murine leukemia virus (MLV) or human immunodeficiency virus (HIV) vectors were used. Retroviruses are indeed well known to be able to incorporate in their envelope a variety of cellular and viral glycoproteins (Ott, 1997; Sandrin et al., 2002). In addition, they can easily package and integrate genetic markers into host cell DNA (Negre et al., 2002). All these properties were exploited to produce viral pseudoparticles expressing E1E2 at their surface and packaging a reporter gene that allows to monitor viral infection of the target cell. HCV pseudoparticles (HCVpp) are produced by transfecting 293T cells with three expression vectors encoding the E1E2 polyprotein, the retroviral core proteins and a packaging-competent retrovirus-derived genome containing a marker gene (Fig. 3). Because MLV and HIV are supposed to assemble at the plasma membrane and HCV glycoproteins are retained in the ER, a first approach has been to modify the transmembrane domains of E1 and E2 to re-address them at the plasma membrane (Hsu et al., 2003; Pohlmann et al., 2003). However pseudoparticles bearing such modified HCV envelope glycoproteins were not infectious. Surprisingly, in the absence of any modification of HCV envelope glycoproteins, infectious

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pseudoparticles were produced (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Interestingly, due to saturation of the ER retention machinery, the cells used to produce HCVpp were shown to express a small fraction of HCV

Fig. 3. Production of HCV pseudoparticules (HCVpp). For the production of HCVpp, human embryo kidney cells 293T are transfected with three expression vectors. The fi rst vector encodes retroviral Gag and Pol proteins. Gag proteins are responsible for particle budding at the plasma membrane and RNA encapsidation via recognition of the specifi c retroviral encapsidation sequence (ψ). The second vector harbors a ψ sequence for encapsidation and encodes a reporter protein (Luciferase). This vector also contains retroviral sequences that are necessary for the reverse transcription of genomic RNA into proviral DNA and for integration of the proviral DNA in the host genomic DNA by the retroviral protein Pol encoded by the fi rst vector. The third vector encodes HCV envelope glycoproteins, which are responsible for the cell tropism and fusion of HCVpp with the target cell membrane. HCVpp contain Gag, Pol, E1 and E2 proteins as well as the RNA encoding the luciferase protein. Infectivity of HCVpp is evaluated by measuring the amount of luciferase expressed in target cells.

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envelope glycoproteins at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). This accumulation at the plasma membrane might therefore be sufficient to incorporate native HCV envelope glycoproteins into retroviral pseudotyped particles.

The data that have been accumulated on these pseudoparticles strongly suggest that they mimic the early steps of HCV infection. Indeed, they exhibit a preferential tropism for hepatic cells and they are specifically neutralized by anti-E2 monoclonal antibodies as well as sera from HCV-infected patients (Bartosch et al., 2003b; Hsu et al., 2003; Op De Beeck et al., 2004). These HCVpp therefore represent the best tool available to study functional HCV envelope glycoproteins. An analysis of the glycoproteins associated with HCVpp has shown the heterogeneous nature of E1 and E2 incorporated into HCVpp (Flint et al., 2004). This highlights the difficulty in identifying forms of the HCV glycoproteins that initiate infection. However, characterization of HCVpp envelope glycoproteins with conformation-sensitive neutralizing monoclonal antibodies has shown that the functional unit is a noncovalent E1E2 heterodimer (Op De Beeck et al., 2004). In addition, coexpression of both envelope glycoproteins has been shown to be necessary to produce infectious pseudoparticles (Bartosch et al., 2003b), confirming that only the E1E2 heterodimer is functional.

HCV RECEPTORSAs a first approach to identify potential HCV receptor(s), a soluble form of HCV glycoprotein E2 has been used. This allowed to identify the CD81 tetraspanin (Levy and Shoham, 2005) as a putative receptor for HCV (Pileri et al., 1998). A very similar approach identified the scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002), a high-density lipoprotein (HDL)-binding molecule (Connelly and Williams, 2004), and the mannose binding lectins DC-SIGN and L-SIGN (van Kooyk and Geijtenbeek, 2003) as additional candidate receptors for HCV (Gardner et al., 2003; Lozach et al., 2003; Pohlmann et al., 2003; Ludwig et al., 2004). Heparan sulfate has also been shown to interact with HCV glycoprotein E2, suggesting that this type of molecule can play a role in HCV entry (Barth et al., 2003). An approach using virus-like particles produced in insect cells has led to the identification of the asialoglycoprotein receptor as another candidate receptor for HCV (Saunier et al., 2003). Finally, because of the physical association of HCV with low- or very-low-density lipoproteins (LDL or VLDL) in serum, the LDL receptor has also been proposed as another candidate receptor for HCV. (Agnello et al., 1999; Monazahian et al., 1999).

A number of cell-surface molecules bind viral envelope glycoproteins without mediating entry, and validation of a viral receptor or co-receptor requires proof that the putative receptor is necessary for infection. This is not easy for HCV due

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to the absence of a robust cell culture system to amplify this virus. The recent development of HCVpp has allowed to further investigate the role of candidate receptors in virus entry. Among all the candidate receptors, only CD81 and SR-BI have been shown to play a direct role in HCVpp entry. Indeed, antibodies directed against CD81 or SR-BI as well as siRNA targeting these receptors reduce HCVpp infectivity (Bartosch et al., 2003b; Bartosch et al., 2003c; Hsu et al., 2003; Cormier et al., 2004b; Zhang et al., 2004a; Lavillette et al., 2005b). A soluble domain of CD81 is also able to compete with HCVpp infectivity (Bartosch et al., 2003b; Hsu et al., 2003). In addition, HDL, the natural ligands of SR-BI, are able to markedly enhance HCVpp entry (Meunier et al., 2005; Voisset et al., 2005). This HDL-mediated enhancement of HCVpp entry involves a complex interplay between SR-BI, HDL and HCV envelope glycoproteins (Voisset et al., 2005). Interestingly, the involvement of CD81 and SR-BI in HCVpp entry seems to be conserved among all the HCV genotypes (McKeating et al., 2004; Lavillette et al., 2005b).

Interactions between viral envelope glycoproteins and potential receptors can have other consequences than virus entry. It has been shown that intracellular interaction between HCV envelope glycoproteins and CD81 can lead to secretion of exosomes containing E1 and E2 glycoproteins (Masciopinto et al., 2004). Interestingly, a soluble form of E2 is also able to bind CD81 at the surface of natural killer cells, and this interaction inhibits cytotoxicity and cytokine production by these cells (Crotta et al., 2002; Tseng and Klimpel, 2002). Binding of a soluble form of E2 can also provide a co-stimulatory signal for T cells (Wack et al., 2001; Soldaini et al., 2003;) and up-regulate matrix metalloproteinase-2 in human hepatic stellate cells (Mazzocca et al., 2005). It remains however to be determined whether HCV glycoprotein expressed in the context of native particles will have the same effects on cell functions.

HCVpp have also been used to investigate the role of other candidate receptors in HCV entry. HCVpp as well as native HCV particles have been shown to bind to cells expressing L-SIGN and DC-SIGN (Gardner et al., 2003; Pohlmann et al., 2003; Lozach et al., 2004). Although these molecules are not expressed on hepatocytes, HCV interactions with L-SIGN and DC-SIGN may contribute to establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell functions as recently suggested (Cormier et al., 2004a; Lozach et al., 2004). Finally, there is no clear evidence that the LDL receptor is a major receptor for HCVpp (Bartosch et al., 2003b).

Interestingly, all the cells permissive to HCVpp co-express CD81 and SR-BI and are of liver origin (Bartosch et al., 2003c; Hsu et al., 2003; Zhang et al., 2004a). However, there are some other cell lines coexpressing CD81 and SR-BI that are non-permissive to infection and which are of non-hepatic origin. These results

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suggest that additional molecule(s), expressed in hepatic cells only, are necessary for HCV entry. Further investigations with HCVpp should allow to identify such molecule(s).

FUNCTIONAL REGIONS OF HCV ENVELOPE GLYCOPROTEINSHCVpp have been used to investigate the functional role of some regions of HCV envelope glycoproteins in virus entry. Mutagenesis studies of the transmembrane domains of HCV envelope glycoproteins have shown that some mutations can affect the entry function of HCVpp without alteration in the biogenesis of E1E2 heterodimer and their incorporation into HCVpp (Ciczora Y, Callens N, Montpellier C, Bartosch B, Cosset FL, Op De Beeck A, Dubuisson J, unpublished data). This suggests that in addition to their role in E1E2 heterodimerization, the transmembrane domains of HCV glycoproteins might play a role in coordinating protein reorganization for the fusion process to occur.

Studies of E2-CD81 interactions and identification of epitopes recognized by antibodies that inhibit these interactions suggest that the CD81-binding region consists of discrete segments of E2 that are rearranged within the same domain during E2 folding (Flint et al., 1999a; Forns et al., 2000a; Yagnik et al., 2000; Owsianka et al., 2001; Clayton et al., 2002; Hsu et al., 2003). Besides this putative binding region, the hypervariable region 1 (HVR1)(Weiner et al., 1991), a 27-amino acid long segment found at the N-terminus of E2 (Fig. 2), has also been suggested to play a role in cell attachment (Penin et al., 2001; Scarselli et al., 2002). This region evolves rapidly in infected individuals, suggesting that it is under strong immune pressure (reviewed in Mondelli et al., 2003). Although an HCV clone lacking HVR1 was shown to be infectious in chimpanzee, this mutant virus was attenuated, suggesting that HVR1 plays a facilitating role in HCV infectivity (Forns et al., 2000b). In addition, deletion of HVR1 reduces HCVpp infectivity (Bartosch et al., 2003c) and abolishes HDL-mediated enhancement of HCVpp infectivity (Voisset et al., 2005). Despite strong amino acid sequence variability related to strong pressure towards change, the chemicophysical properties and conformation of HVR1 are highly conserved, and HVR1 is a globally basic stretch, with basic residues located at specific sequence positions. Functional studies of HCVpp containing mutations in HVR1 indicate that infectivity increases with the number of basic residues in HVR1 (Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J, unpublished data). In addition, a shift in position of some charged residues modulates infectivity. These data suggest that HVR1 is a region involved in interaction with a host molecule involved in HCV entry. However, it remains to be determined whether SR-BI or another putative receptor is involved in this interaction.

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HCV envelope glycoproteins are highly glycosylated and some maturation of these glycans has been observed on HCV envelope glycoproteins associated with HCVpp (Flint et al., 2004; Lozach et al., 2004; Op De Beeck et al., 2004). Mutation of some glycosylation sites in HCV envelope glycoproteins can reduce or abolish HCVpp infectivity without apparently affecting folding and incorporation of the glycoproteins into the particles (Goffard et al., 2005). N-linked glycans at position N2 and N4 of E2 have indeed been shown to be essential for the entry functions of HCV envelope glycoproteins (Fig. 2). In addition, some other glycans (N2 of E1 and N5, N6 and N11 of E2) can also modulate HCVpp entry. Further studies will be necessary to determine whether these mutations affect receptor binding or the fusion properties of HCV envelope glycoproteins.

MECHANISMS OF HCV ENTRYVirus attachment to receptors initiates a series of events that lead to virus entry. For enveloped viruses, the entry process is controlled by viral surface glycoproteins that undergo triggered conformational changes from a metastable state to a lower energy state. This structural change leads to the exposure of a buried functional element, named the fusion peptide and is believed to provide the energy required for the merging of the lipid bilayers (reviewed in Colman and Lawrence, 2003). So far, viral fusion proteins have been shown to fall into two different structural classes designated as class I and II (reviewed in Earp et al., 2005). Class I fusion proteins possess N-terminal or N-proximal fusion peptides, and they are synthesized as a precursor that is cleaved into two subunits by host cell proteases. In some cases (e.g., influenza HA), the two subunits remain associated through a disulfide bond, whereas in others (e.g. HIV Env) the two subunits remain associated through noncovalent interactions. The proteolytic processing event creates the metastable state of the fusion protein (Colman and Lawrence, 2003). In their native metastable conformation, class I fusion proteins form trimeric spikes at the surface of the virions with the fusion subunit being highly helical. Upon a fusion trigger event (receptor binding at the cell surface or low pH in endosomes), the trimeric proteins transiently form an extended conformation allowing the hydrophobic fusion peptide to insert into the target membrane. Protein refolding leads then to the formation of very stable trimeric structures in which both the N-proximal fusion peptide and the C-proximal membrane anchor are juxtaposed at the same end to allow virus and cell membrane connection and hemifusion (reviewed in Colman and Lawrence, 2003).

Class II viral fusion proteins have a completely different structure. They are predominantly non-helical, instead having a beta-sheet type structure; they are not cleaved during biosynthesis; and they possess an internal fusion peptide with a loop conformation (reviewed in Earp et al., 2005). The proteins are oriented parallel to the membrane, and they have a three-domain architecture with domain I beginning at the N-terminus, domain II containing the internal fusion loop, and domain III

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being at the C-terminus. In addition, class II fusion proteins are synthesized as a complex with a second membrane glycoprotein (prM for fl aviviruses; pE2 for alphaviruses). Newly synthesized E and prM proteins of the tick borne encephalitis virus associate to form noncovalent heterodimers (Fig. 4) that are incorporated into immature virions by budding into the ER lumen (Allison et al., 1995; Mackenzie and Westaway, 2001). These particles are then transported through the secretory pathway and shortly before release from the cell, the activation of the fusogenic potential occurs by the cleavage of the accessory protein prM by a cellular furin protease in the trans-Golgi network (Stadler et al., 1997). After prM cleavage, the E protein exists as a metastable homodimers at the virion surface. The ectodomains of the dimers are orientated antiparallel to one another (Rey et al., 1995; Lescar et al., 2001; Modis et al., 2003). The architecture of the alphavirus Semliki Forest virus spike is similar to that of tick borne encephalitis virus E, but in this case, the metastable oligomer is a heterodimer of the fusion protein E1 and the companion protein E2 with an associated small protein E3 (Lescar et al., 2001). In addition,

Fig. 4. Comparison of fl avivirus and hepacivirus envelope proteins. In the Flaviviridae family, class II fusion proteins (depicted in light grey) have been described in the fl aviviruses (E protein of tick born encephalitis and dengue viruses). They are synthesized as a complex with a second membrane glycoprotein (depicted in dark grey). Shortly before release from the cell, activation of the fusogenic potential occurs by cleavage of the accessory protein (arrow). HCV envelope glycoproteins are supposed to belong to the class II fusion proteins, but contrary to fl aviviruses, HCV envelope proteins are highly glycosylated and are not matured by a cellular endoprotease during their transport through the secretory pathway.

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contrary to flaviviruses, alphaviruses have been shown to bud from the plasma membrane.

Both alphaviruses and flaviviruses enter target cells by receptor-mediated endocytosis. The receptor recognition function is carried by the fusion protein itself for the flaviviruses (E) and by the companion protein (E2) for the alphaviruses. Exposure to the acidic pH of the endosomes triggers a major conformational change of the envelope involving dissociation of the native homodimer (for flaviviruses) or heterodimer (for alphaviruses) and the irreversible formation of homotrimers of the fusion proteins (Earp et al., 2005; Mukhopadhyay et al., 2005).

Based on its classification in the Flaviviridae family, HCV envelope has been proposed to contain a class II fusion protein (Yagnik et al., 2000). As found in the case of alphaviruses and flaviruses, HCVpp entry is pH dependent (Bartosch et al., 2003c; Hsu et al., 2003). These observations indicate that HCV may enter the cells through endocytosis. The cell surface receptor(s) recognized by HCV should therefore traffic cell-bound virions to endosomal compartments. However, characterization of the route of HCV entry needs further investigations. Contrary to what is observed for other class II envelope proteins, there is no evidence that HCV envelope glycoproteins are matured by a cellular endoprotease during their transport through the secretory pathway (Op De Beeck et al., 2004). In addition, HCV envelope glycoproteins are highly glycosylated, whereas other described class II envelope proteins contain a very low number of glycans (Fig. 4). Interestingly, some of the glycans present on HCV envelope glycoproteins seem to be involved in controlling HCV entry (Goffard et al., 2005).

There remains some controversy on the identity of HCV fusion protein. It has been proposed that E1 might be a good candidate because sequence analyses suggest that it might contain a putative fusion peptide in its ectodomain (Flint et al., 1999b; Garry and Dash, 2003). On the other hand, potential structural homology with other class II fusion proteins suggests that E2 could be the fusion protein (Yagnik et al., 2000). Mutagenesis studies in the putative fusion peptides of the envelope glycoproteins associated with HCVpp as described for the flavivirus envelope protein E (Allison et al., 2001), should be helpful for further characterization of HCV fusion protein. In addition, a high-resolution structure of HCV envelope glycoproteins would also help understanding the fusion mechanism of the virus.

INHIBITION OF HCV ENVELOPE GLYCOPROTEIN FUNCTIONS BY NEUTRALIZING ANTIBODIESBecause they are exposed at the surface of the virion, the envelope proteins are targets of neutralizing antibodies. These antibodies block a viral infection by inhibiting virion binding or membrane fusion. Understanding the mechanisms of neutralization needs therefore a good knowledge of the mechanism of entry. The

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role of neutralizing antibodies in HCV infection and disease progression remained unclear for a long time, largely because of the lack of assays to measure and quantify their activity. Previous experiments showed that serum from a chronically infected patient could neutralize HCV infectivity in a chimpanzee model, giving evidence for antibody-mediated neutralization of HCV (Farci et al., 1994). Neutralizing antibodies could also be identified by their ability to prevent HCV replication in a lymphoid cell line (Shimizu et al., 1994; Shimizu et al., 1996).

The recent development of HCVpp offers the possibility to study HCV neutralization with defined sequences of HCV envelope glycoproteins, and the use of HCVpp in neutralization studies has been validated (Bartosch et al., 2003a). As determined with HCVpp, it seems that the majority of chronically infected patients have cross-reactive neutralizing antibodies (Logvinoff et al., 2004; Meunier et al., 2005). In contrast, neutralizing antibodies have not been detected in several cases of acute resolving infection (Logvinoff et al., 2004; Meunier et al., 2005), and the detection of neutralizing antibodies in acutely infected individuals did not seem to be associated with viral clearance (Logvinoff et al., 2004). However, another study has shown in some patients a progressive emergence of a relatively strong neutralizing response in correlation with a decrease in viremia (Lavillette et al., 2005a). Further investigations on a large number of acutely infected patients will be necessary to determine the role of neutralizing antibodies in controlling HCV infections. Interestingly, it has been observed that HCVpp infectivity is enhanced by human sera, and this enhancement of infectivity can partly mask the presence of neutralizing antibodies (Lavillette et al., 2005a; Meunier et al., 2005). In addition, HDL have been identified as the component responsible for serum-mediated enhancement of infectivity (Meunier et al., 2005; Voisset et al., 2005).

For a long time, the HVR1 sequence of E2 has been proposed to be a major target for neutralizing antibodies (Kato et al., 1993; Farci et al., 1996). However, data obtained with the HCVpp model indicate that neutralizing epitopes located outside of HVR1 also exist (Bartosch et al., 2003a). Interestingly, characterization of HCVpp with monoclonal antibodies has allowed to identify conformation-dependent and -independent neutralizing epitopes outside of HVR1 (Fig. 2)(Bartosch et al., 2003b; Hsu et al., 2003; Keck et al., 2004; Op De Beeck et al., 2004). Conformation-dependent human monoclonal antibodies have also allowed to identify three immunogenic domains in E2 with neutralizing antibodies being restricted to two of these domains (Keck et al., 2004). Whether E2 domains identified with these monoclonal antibodies are similar to the antigenic structural and functional domains of the envelope protein E of the flaviviruses (Rey et al., 1995) remains to be determined.

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CONCLUSIONStudies of the biogenesis of HCV envelope glycoproteins have shown the pivotal role of the transmembrane domains in the assembly of a noncovalent E1E2 heterodimer in the ER. More recently, the development of the HCVpp model has allowed to investigate the role of E1E2 heterodimer in virus entry. Functional regions in HCV envelope glycoproteins can now be identified and potential receptors can also be validated. Entry is an essential step in the life cycle of a virus, which can potentially be blocked by neutralizing antibodies or antiviral drugs that target the envelope proteins of the virus. Understanding the viral and cellular components involved in HCV invasion into the host cell, combined with a comprehension of the mechanisms that govern this process, should therefore open the possibility of developing new therapeutic approaches.

FUTURE TRENDSThe development of the HCVpp model has allowed to initiate the characterization of the entry function of HCV envelope glycoproteins. The use of HCVpp will continue to provide additional information on the role of HCV envelope glycoproteins in viral entry. However, the recent development of a full-length clone that is infectious in cell culture (see chapter 16) provides new opportunities to study the functions of HCV envelope glycoproteins. A comparison of the properties of HCV envelope glycoproteins produced in HCVpp and in this infectious clone will be very useful to validate the data that have been generated during the past three years. In addition, this infectious clone will allow for the first time to decipher the role of HCV envelope glycoproteins in virion assembly. Finally, obtaining a high-resolution structure of HCV envelope glycoproteins will also be necessary to understand the fusion mechanism of this virus.

ACKNOWLEDGMENTSWe thank Sophana Ung for preparing the illustrations. Our research was supported by EU grant QLRT-2000-01120 and QLRT-2001-01329 and grants from the "Agence Nationale de Recherche sur le Sida et les hépatites virales" (ANRS), INSERM "ATC-Hépatite C" and the "Association pour la Recherche sur le Cancer" (ARC).

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HCV NS2/3 Protease

Chapter 5

HCV NS2/3 Protease

Sarah Welbourn and Arnim Pause

ABSTRACTThe hepatitis C virus NS2/3 protein is a highly hydrophobic protease responsible for the cleavage of the viral polypeptide between non-structural proteins NS2 and NS3. However, many aspects of the NS2/3 protease's role in the viral life cycle and mechanism of action remain unknown or controversial. NS2/3 has been proposed to function as either a cysteine or metalloprotease despite its lack of sequence homology to proteases of known function. In addition, although shown to be required for persistent infection in a chimpanzee, the role of NS2/3 cleavage in the viral life cycle has not yet been fully investigated due to the lack of an in vitro system in which to study all aspects of HCV replication. However, several recent studies are beginning to clarify possible roles of the cleaved NS2 protein in modulation of host cell gene expression and apoptosis.

INTRODUCTIONThe NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. Extending from amino acids 810-1206, NS2/3 is the first non-structural (NS) protein translated and is responsible for the intramolecular cleavage between NS2 and NS3 (see Fig. 1). The amino terminus of NS2 is cleaved from the adjacent p7 protein by host signal peptidases in a membrane-dependent

C E1 E2 P7 NS2 NS3 NS4A NS5A NS5BNS4B

NS2 NS3

810 1206NS2/3

810 120610271026

Fig. 1. The HCV NS2/3 protease. The NS2/3 protease is shown in the context of the HCV polyprotein. NS2 and the protease domain of NS3 (from aa 810 to 1206) constitute NS2/3, which undergoes autocatalytic cleavage between aa 1026 and 1027.

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manner, while the chymotrypsin-like serine protease located in NS3 is responsible for the cleavage at the NS3/4A and downstream junctions. The HCV NS2/3 protein is an autoprotease whose activity is separate from NS3 protease functions (see Chapter 6). Although many studies have focused on the residues and sequences required for efficient NS2/3 processing, the exact nature of the protease has still not been firmly established, with it being proposed to function as either a novel cysteine or metalloprotease. Furthermore, although all NS proteins are proposed to play a role in viral replication, the exact functions of HCV NS2/3, as well as cleaved NS2 remain largely unexplored; however, some interesting potential functions have emerged in recent years. This chapter will focus on the known properties of the NS2/3 protease as well as the possible functions of both the NS2/3 protease and the NS2 protein.

NS2/3 CATALYTIC CLEAVAGEGENERAL STRUCTURAL FEATURES OF NS2/3The NS2/3 protease is responsible for the intramolecular cleavage of NS2 from NS3 between aa 1026 and 1027 (Grakoui et al., 1993; Hijikata et al., 1993a). Fig. 2 shows the main structural and functional domains of the protein. NS2 contains a highly hydrophobic N-terminal region suggested to contain multiple transmembrane segments; however, this region is not required for efficient cleavage at the NS2/3 site (Hijikata et al., 1993a; Pallaoro et al., 2001; Thibeault et al., 2001). The minimal domain for activity of the enzyme has been mapped to aa 907-1206 (Pallaoro et al., 2001). This encompasses the C-terminal portion of NS2, immediately following the hydrophobic region, as well as the N-terminal protease domain of NS3. Although these sequences are required and sufficient for cleavage activity, processing is not dependent the NS3 serine protease activity (Grakoui et al., 1993; Hijikata et al., 1993a). This differs from the NS2B protein of flaviviruses, in which the NS3

Hydrophobic Region

810

NS3NS2

Minimal Region for NS2/3 activity

1206

* * * *

C1123 C1125C1171 H1175

NS3 Structural Zinc Binding Sites

* *H952 C993

1026

*E972

907

Fig. 2. Functional domains of the NS2/3 protease. The NS2/3 protease encompasses an N-terminal hydrophobic region, with a minimal domain required for activity between aa 907 and 1206. Residues in NS2 required for NS2/3 processing (H942, E972, C993), as well as residues in NS3 responsible for the coordination of a zinc atom are shown.

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protease performs a cis-cleavage at the NS2B site and then uses NS2B as a co-factor for the processing of the downstream NS polypeptide (Chambers et al., 1991; Chambers et al., 1990; Falgout et al., 1991).

NS2/3 PROCESSING REQUIREMENTSThe HCV NS2/3 protease shows no sequence motifs typical of known proteases; however, sequence alignments show similarity with the GBV NS2/3 protein as well as the bovine viral diarrhea virus (BVDV) NS2/3 protein (Lackner et al., 2004). Residues H952, E972 and C993 are conserved among all genotypes of HCV and mutation of H952 or C993 to alanine completely inhibits NS2/3 cleavage activity while a glutamic acid 972 to glutamine substitution also significantly affects processing (Grakoui et al., 1993; Hijikata et al., 1993a). Furthermore, although NS3 serine protease activity is not required for NS2/3 processing, the full NS3 protease domain must be present and cannot be substituted for another NS protein (Santolini et al., 1995). In addition, mutation of cysteine residues 1123, 1127 and 1171 in NS3, which together with H1175 participate in the coordination of a zinc molecule (Kim et al., 1996; Love et al., 1996), abolishes both NS3 and NS2/3 activities (Hijikata et al., 1993a), presumably by disrupting folding of the enzymes. This therefore suggests that the NS3 protease domain is required to play a structural role in the folding of the enzyme.

Proper folding of the NS2/3 protein and cleavage site plays an important role in the efficiency of NS2/3 processing. Residues surrounding the cleavage site, WRLL↓APIT, are highly conserved between HCV genotypes but are remarkably resistant to mutations (Hirowatari et al., 1993; Reed et al., 1995). Only mutations severely affecting the conformation of the cleavage site (such as deletion or proline substitution of P1 or P1') severely inhibit cleavage. Furthermore, NS4A-derived peptides that upon binding cause a conformational rearrangement of the NS3 N-terminus are potent inhibitors of NS2/3 activity, likely by altering the positioning of the cleavage site (Darke et al., 1999; Thibeault et al., 2001). The presence of microsomal membranes or non-ionic detergents has been found to be required for in vitro processing at the NS2/3 site in certain genotypes (Pieroni et al., 1997; Santolini et al., 1995), while increasing the efficiency of cleavage of others (Grakoui et al., 1993; Santolini et al., 1995), suggesting the hydrophobic environment is necessary for proper folding of the enzyme and positioning of the cleavage site. Similarly, Waxman et al. (2001) have demonstrated the requirement for the ATP hydrolyzing ability of molecular chaperone HSP90 for efficient cleavage in in vitro and cell based assays. A similar phenomenon has been described for the BVDV NS2/3 protein where a cellular DnaJ chaperone protein, Jiv, has been found to associate with and modulate NS2/3 activity, possibly by causing a conformational change in the protein (Rinck et al., 2001). Although the mechanisms are still unclear, this could point to a role of cellular chaperones in inducing/maintaining the proper conformation of NS2/3 required for cleavage.

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MECHANISM OF ACTION: CYSTEINE OR METALLOPROTEASE?Initial studies showing NS2/3 activity is inhibited by EDTA and stimulated by zinc led to the early suggestion that NS2/3 functions as a zinc-dependent metalloprotease (Hijikata et al., 1993a). However, with the discovery of the importance of zinc for the structural integrity of the NS3 protease domain, others have proposed NS2/3 may be a novel cysteine protease with a catalytic dyad comprised of H952 and C993 with the possible involvement of E972 as the third residue of a catalytic triad. Inhibition studies both in in vitro translation systems and with purified proteins have failed to yield a definite classification (Pallaoro et al., 2001; Pieroni et al., 1997; Thibeault et al., 2001). Although inhibited by metal chelators such as phenanthroline and EDTA, this inhibition is relieved by the addition of ZnCl2, CdCl2 or MgCl2. This could therefore point to a structural rather than catalytic role for the zinc molecule as Cd has not traditionally been able to functionally replace Zn in other metalloproteases (Angleton and Van Wart, 1988; Cha et al., 1996; Holland et al., 1995). However, although classical cysteine protease inhibitors iodoacetamide and N-ethylmaleimide show strong inhibition of NS2/3 processing, no single cysteine has been found to be more susceptible to these alkylating agents (Pallaoro et al., 2001).

Recently, conserved His, Cys and Glu have also been found to be present in BVDV strains and required for NS2/3 cleavage in vitro (Lackner et al., 2004), suggesting a similar mechanism of action of the two proteases. However, several differences exist. In addition to the necessity of the N-terminal hydrophobic region of NS2, BVDV NS2/3 does not require the full NS3 protease domain for activity, but rather possesses a conserved zinc-binding site within NS2 itself (Lackner et al., 2004). Although no traditional metal-binding sequences have been identified in HCV NS2, the presence of an additional catalytic zinc in NS2 or a catalytic role for the NS3 zinc molecule cannot be definitely ruled out. The elucidation of the so far unknown crystal structure of NS2/3 should bring important insights into the mechanism of cleavage of this enzyme.

NS2/3 BIMOLECULAR CLEAVAGEBimolecular cleavage of NS2/3 has been shown to occur, albeit inefficiently, in cell transfection experiments (Grakoui et al., 1993; Reed et al., 1995). In this system, NS2/3 proteins with mutations/deletions in either the NS2 or NS3 domains could support cleavage provided the missing functional region was co-expressed on a separate polypeptide. In addition, catalytically inactive NS2/3 mutants were also found to inhibit processing of a wild-type protein when expressed in trans. The observation that a recombinant NS2/3 protein forms dimers in vitro is consistent with these findings (Pallaoro et al., 2001). However, no trans cleavage has been observed using purified proteins (Pallaoro et al., 2001; Thibeault et al., 2001). Interestingly, NS2/3 activity in vitro was found to be concentration dependent, supporting the notion that dimer formation is essential for the reaction (Pallaoro et al., 2001). Dimitrova et al. (2003) have also demonstrated the homo-association of

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the NS2 protein in various systems and suggest that the cleavage between NS2 and NS3 could potentially be performed by dimers of NS2/3 encoded on neighbouring polyprotein chains. As NS2/3 cleavage is widely believed to be an intramolecular event, the significance of bimolecular cleavage in the polyprotein processing events of HCV infection in vivo remains to be determined.

ROLE OF NS2/3 CLEAVAGE IN VIRAL REPLICATIONThe role of the NS2/3 protease in HCV replication remains to be fully understood. NS2/3 cleavage is required for viral replication in vivo, as demonstrated by an HCV clone devoid of NS2/3 activity that fails to cause a persistent infection in a chimpanzee (Kolykhalov et al., 2000). However, NS3-3' UTR subgenomic replicons not encoding the NS2 protein replicate efficiently in Huh-7 cells (Lohmann et al., 1999), suggesting NS2/3 is not strictly required for genome replication.

If cleavage at the NS2/3 site occurs solely for the release of the NS2 protein, what is the advantage for the virus of encoding two distinct proteases for polyprotein processing? Although several roles have been proposed for the cleaved NS2 protein, the NS2/3 protease itself appears unique in that its activity subsequently causes its inactivation. However, potential regulation of the cleavage reaction could have other implications for the viral life cycle, as is known for BVDV NS2/3 processing. BVDV stains are present in two forms, non-cytopathic (noncp) which expresses primarily uncleaved NS2/3 and has the ability to cause persistent infection and cytopathic (cp) strains expressing cleaved NS3 (Donis and Dubovi, 1987; Pocock et al., 1987). For this pestivirus, RNA replication levels have been shown to correlate with amount of cleaved NS3 protein (Lackner et al., 2004), whereas the uncleaved NS2/3 is required for viral infectivity (Agapov et al., 2004). Evolution of a cp strain from a non-cp strain occurs through the activation of the NS2/3 cleavage by a variety of mutations, deletions, duplications and rearrangements within the NS2 region (Kummerer et al., 1998; Meyers et al., 1992; Tautz et al., 1996; Tautz et al., 1994). However, it has recently been suggested that BVDV NS2/3 is an autoprotease whose temporal regulation is involved in modulating the different stages of RNA replication and viral morphogenesis (Lackner et al., 2004). Whether HCV NS2/3 could perform a similar regulatory role remains to be determined. Although NS2/3 processing appears to be a very efficient event in cell expression systems, the possible role for an uncleaved NS2/3 precursor in the complete viral life cycle has not been ruled out..NS2 AS PART OF THE REPLICATION COMPLEX?HCV RNA replication has been proposed to occur via the formation of a membrane bound replication complex that comprises the association of the NS proteins required for genome replication (NS3-5B). However, due to the lack of an efficient cell culture system to study the viral life cycle, studies focusing on the

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replication complex have been so far limited to the subgenomic replicon system (see Chapter 11), where NS2 is not expressed. Several studies have indicated that NS2 is an integral membrane protein that is targeted to the endoplasmic reticulum (ER) (Santolini et al., 1995; Yamaga and Ou, 2002). Interestingly, NS2 has been found by one group to be inserted into the membrane only when expressed in the context of the NS2/3 protein, and only after cleavage from NS3 (Santolini et al., 1995). NS2 has also been found to interact with all other HCV NS proteins in in vitro pull-down, as well as cell-based co-localization and co-immunoprecipitation experiments (Dimitrova et al., 2003; Hijikata et al., 1993b). Therefore, although not required for RNA replication, the possible presence of NS2 in this complex as an accessory protein is plausible and warrants further investigation.

ROLES OF CLEAVED NS2

HCV NS2 IS AN INTEGRAL MEMBRANE PROTEINThe NS2 protein derived from the cleavage of NS2/3 is inserted into the ER membrane through its N-terminal hydrophobic domain. However, the exact mechanisms of translocation as well as the membrane topology of the protein remain controversial. Membrane association has been found to be dependent on SRP-SRP receptor targeting (Santolini et al., 1995). It was originally proposed that a signal sequence present in upstream p7 was required for membrane association co-translationally, although NS2 translocation has subsequently been demonstrated by several groups to be p7 independent (Santolini et al., 1995; Yamaga and Ou, 2002). Furthermore, although the cleavage at the p7-NS2 junction is performed in a membrane-dependent fashion by signal peptidase (Lin et al., 1994; Mizushima et al., 1994) and the presence of membranes is stimulatory (and for some strains required) for NS2/3 cleavage, one group has shown that the integration of NS2 into the membrane is performed post-translationally, and only after cleavage from NS3 (Santolini et al., 1995). However, Yamaga and Ou (2002) have since proposed that translocation could occur co-translationally and therefore the exact mechanisms of integration remain unclear. The amino terminal region of NS2 is likely to span the membrane several times (Pallaoro et al., 2001; Yamaga and Ou, 2002). However, the exact number of transmembrane domains, as well as the orientation of the protein in the membrane have not been conclusively determined.

NS2 AND NS5A HYPERPHOSPHORYLATIONHCV NS5A has many roles in both RNA replication and the modulation of the host cell environment during infection and has been found to be present in two distinct phosphorylated forms: p56 and p58 (see Chapter 9). Liu et al. have reported the importance of NS2 for the generation of hyperphosphorylated NS5A (p58) (Liu et al., 1999). Using plasmids expressing various sections of the HCV polyprotein in transient transfection experiments, they demonstrate the requirement of NS2 generated by the cleavage of NS2/3 for the formation of p58. However, while

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performing similar experiments, other groups have demonstrated the appearance of p58 without the presence of NS2 (Koch and Bartenschlager, 1999; Neddermann et al., 1999). Indeed, Neddermann et al. (1999) therefore suggested that NS2 itself is not required for the hyperphosphorylation process, but rather that it could be the authentic N-terminus of NS3, generated by NS2/3 cleavage, that is of importance.

NS2 INHIBITION OF GENE EXPRESSIONNS2 may also play a role in modulating cellular gene expression in infected cells. One study by Dumoulin et al. (2003) found that NS2 exerted a general inhibitory effect on the expression of a reporter gene expressed from a variety of different promoters (human ferrochelatase promoter, NFkappaB binding sites, SV40 promoter/enhancer sequences, full length, as well as minimal TNF-alpha promoters and cytomegalovirus immediate-early promoter) in several different hepatic and non-hepatic cell types. The amino-terminal (810-940) region of NS2 was sufficient to cause this effect, suggesting inhibition of gene expression is not dependent on the activity of the NS2/3 protease itself. It was therefore suggested that NS2 could potentially regulate host cell protein levels by interfering with a general aspect of transcription or translation. Indeed, several other HCV-encoded proteins, including core (Chapter 3), NS4B (Chapter 8) and NS5A (Chapter 9), have been demonstrated to alter cellular gene expression though a variety of mechanisms (Kato et al., 1997; Kato et al., 2000; Naganuma et al., 2000; Ray et al., 1995). This aspect of NS2 function will require further confirmation and careful investigation as it indicates a potential role for NS2 in the modulation of the host cell environment which has important implications for both the establishment of persistent infection and the pathogenesis of chronic HCV.

NS2 AND APOPTOSISIn order to establish a persistent infection, many viruses have evolved mechanisms to interfere with cellular apoptosis. In this manner, the virus is then able to replicate to sufficient levels without the elimination of the host cell. Several HCV proteins have been implicated in the modulation of cell signalling and apoptosis, including core, E2, NS5A and NS2 (Gale et al., 1997; Honda et al., 2000; Machida et al., 2001; Ruggieri et al., 1997). Machida et al. (2001) have reported that Fas-mediated apoptosis is inhibited in transgenic mice expressing HCV core, E1, E2 and NS2 proteins. The expression of these proteins in the liver prevented cytochrome c release from the mitochondria as well as preventing the activation of caspase 9 and caspase 3/7, but did not affect caspase 8. Therefore, this implicates these HCV proteins in the mitochondrial intrinsic apoptotic pathway, which involves mitochondrial membrane permeabilization and the release of pro-apoptotic factors, resulting in cell death. Furthermore, Erdtmann et al. (2003) showed that NS2 inhibits CIDE-B-induced apoptosis in co-expression experiments. CIDE-B (cell death-inducing DFF45-like effector) is a mitochondrial pro-apoptotic protein whose overexpression has

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been shown to induce cell death (Inohara et al., 1998). CIDE-B-induced apoptosis requires mitochondrial localization and dimerization of the protein, both of which are mediated by a region in its C-terminal domain (Chen et al., 2000). NS2 was found to interact specifically with the C-terminal region of CIDE-B and block cytochrome c release from the mitochondria as well as cell death (Erdtmann et al., 2003). NS2 could therefore potentially prevent the dimerization of CIDE-B required for activity. However, the mechanism of inhibition remains unclear as NS2 is thought to be localized at the ER membrane. In this case, NS2 could potentially bind and sequester CIDE-B, preventing its localization at the mitochondria.

The roles of mature cleaved NS2 remain largely unexplored. Although some possible functions have been proposed and are described here, the lack of an efficient cell culture system remains a major hurdle in identifying the main tasks of NS2 in the various events of the viral life cycle. Furthermore, it has been observed that NS2 is a short-lived protein in replicon cells (Franck et al., 2005). Franck et al. (2005) showed that NS2 is a target for phosphorylation by CK2 and is subsequently rapidly degraded by the proteasome. This appears to be a ubiquitin-independent process and the exact mechanisms involved have yet to be identified. However, the regulation of this process could have important implications for the understanding of the various functions of NS2 and the sequential events of the viral life cycle.

CONCLUSIONSMuch work is still required in the study of the NS2/3 protease. Although several studies over the past decade have focussed on NS2/3 cleavage, the catalytic mechanism of the enzyme remains controversial. Initial attempts at characterizing the enzyme were limited to in vitro and cell expression systems and despite the development in recent years of in vitro systems in which the processing reaction can be studied using purified recombinant proteins, a definitive classification has not yet been determined. A three dimensional structure of NS2/3 is very much needed and will likely yield important insights into the mechanism of action of the enzyme.

Similarly, a robust cell culture system for the study of the viral life cycle is of urgent need (see Chapter 16). Such a system will be crucial to precisely define the roles of NS2/3 cleavage and the NS2 protein in the complete viral life cycle. Of particular interest are the observations that NS2 could potentially modulate the host cell environment during HCV infection through interference with gene expression and cellular apoptosis. However, it will be necessary to validate these findings in a more physiologically relevant setting.

Although its mode of action is unclear, NS2/3 cleavage is absolutely required for persistent viral infection in a chimpanzee. The HCV NS2/3 protease shares no obvious sequence homology to any known proteases in the animal kingdom and

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would therefore make an attractive target for antiviral therapy. The elucidation of the crystal structure of NS2/3, its mechanism of action and precise functions in replication will help to generate important information for the development of strategies for inhibition of NS2/3 processing, which could become the basis for novel HCV therapies in the future.

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Pieroni, L., Santolini, E., Fipaldini, C., Pacini, L., Migliaccio, G., and La Monica, N. (1997). In vitro study of the NS2-3 protease of hepatitis C virus. J Virol 71, 6373-6380.

Pocock, D. H., Howard, C. J., Clarke, M. C., and Brownlie, J. (1987). Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus. Arch Virol 94, 43-53.

Ray, R. B., Lagging, L. M., Meyer, K., Steele, R., and Ray, R. (1995). Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 37, 209-220.

Reed, K. E., Grakoui, A., and Rice, C. M. (1995). Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage. J Virol 69, 4127-4136.

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HCV NS3-4A Serine Protease

Chapter 6


Chao Lin

ABSTRACTThe 9.6 kb plus-strand RNA genome of HCV encodes a long polyprotein precursor of ~3,000 amino acids, which is processed by cellular and viral proteases to 10 individual proteins. One of the HCV proteases, NS3-4A serine protease, is a non-covalent heterodimer consisting of a catalytic subunit (the N-terminal one-third of NS3 protein) and an activating cofactor (NS4A protein), and is responsible for cleavage at four sites of the HCV polyprotein. HCV NS3-4A protease is essential for viral replication in cell culture and in chimpanzees, and has been considered as one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of small-molecule, selective inhibitors against HCV NS3-4A protease as oral drug candidates has been hampered by its shallow substrate-binding groove and the lack of robust, reproducible viral replication models in cell culture or in small animals. Nevertheless, decade-long intense efforts by many groups have largely overcome these two obstacles and provided fruitful understanding of its biological functions, biochemistry, and three-dimensional structures, culminating in recent demonstration of proof-of-concept anti-HCV activities in patients. This chapter will review key findings in these areas, and focus on the discovery and clinical development of HCV NS3-4A protease inhibitors as novel antiviral therapies.

INTRODUCTIONThe hepatitis C virus (HCV) epidemic, affecting ~170 million people worldwide, has been widely discussed (Memon and Memon, 2002; Wasley and Alter, 2000). The current standard therapy for chronic hepatitis C patients is a combination of weekly injections of pegylated interferon (IFN)-α, and daily oral doses of ribavirin (for a review, see Anonymous, 2002; Strader et al., 2004 and references therein). Both drugs are indirect antivirals because they do not target a specific HCV protein or RNA element. A sustained viral response (SVR), which is defined as treated patients remaining HCV-free (undetectable viral load) for 6 months after the termination of therapy, is achieved in only half of the treated patients and in less than half of patients with genotype 1 HCV or with high viral load (Fried et al., 2002; Hadziyannis et al., 2004; Manns et al., 2001). The standard therapy is associated with considerable adverse effects, including depression, fatigue, and "flu-like" symptoms caused by IFN-α, and hemolytic anemia by ribavirin. There is a huge unmet medical need

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for orally available, small-molecule, direct anti-HCV drugs to provide hepatitis C patients more effective treatments with fewer side effects.

HCV, a member of the Flaviviridae family of viruses, has a 9.6 kb plus-strand RNA genome that encodes a long polyprotein precursor of ~3,000 amino acids, which is processed proteolytically upon translation by both cellular and viral proteases to at least 10 individual proteins, including four structural proteins (C, E1, E2 and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Fig. 1) (for a review see Lindenbach and Rice, 2001). The NS3 protein is a multi-functional protein, with a serine protease domain in its N-terminal one-third and a helicase domain in the C-terminal two-third (reviewed in chapter 7). The NS3-4A serine protease is a non-covalent, heterodimer complex formed by two HCV-encoded proteins, the N-terminal serine protease domain of NS3 (catalytic subunit) and the NS4A cofactor (activation subunit). The NS3-4A serine protease is responsible for the proteolytic cleavage at four junctions of the HCV polyprotein precursor: NS3/NS4A (self cleavage), NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B (Fig. 1) (Bartenschlager et al., 1993; Bartenschlager et al., 1995b; Failla et al., 1995; Grakoui et al., 1993a; Grakoui et al., 1993b; Hijikata et al., 1993b; Kim et al., 1996; Lin and Rice, 1995; Lin et al., 1995; Tanji et al., 1995; Tomei et al., 1993). HCV encodes four viral enzymes in its nonstructural protein region: NS2-3 autoprotease (reviewed in chapter 5) and NS3-4A serine protease (reviewed in this chapter), NS3 helicase (reviewed in chapter 7) and NS5B RNA-depdendent RNA polymerase (reviewed in chapter 10), all of which are essential for HCV replication or infectivity in chimpanzees (Kolykhalov et al., 2000). Among them, NS3-4A serine protease and NS5B RNA-dependent RNA polymerase are generally considered to be the most attractive targets for design of new anti-HCV oral drugs.

The success of HIV protease inhibitor drugs demonstrates that viral proteases, such as the HCV NS3-4A protease, could be excellent targets for a structure-based drug design approach. However, the shallow substrate-binding groove of the HCV

Fig. 1. A schematic diagram of the HCV genome. The 5' and 3' untranslated regions (UTR) are shown with putative secondary structures. The polyprotein encoded by the long open reading frame is shown as a long box, in which individual mature protein products are labeled as core (C), envelope proteins 1 and 2 (E1 and E2), p7, followed by six nonstructural proteins (NS) 2, 3, 4A, 4B, 5A, and 5B. The cleavage sites are marked for cellular signal peptidase (filled triangle), HCV NS2-3 auto-protease (filled arrow), and NS3-4A serine protease (open arrow).

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NS3-4A serine protease observed in an X-ray crystal structure (Kim et al., 1996) suggested that discovery of a potent, small-molecule, and orally available drug candidate would be an enormously challenging task. Despite of the lack of a robust and consistent HCV infection cell culture, a subgenomic replicon system developed by Lohmann et al. (1999) became the workhorse as the standard assay of antiviral activity of the HCV NS3-4A protease inhibitors. In addition, the lack of a robust HCV infection model in small animals has generally forced scientists to rely on a combination of anti-HCV activity in cell culture and animal pharmacokinetics as surrogate indicators of efficacy prior to clinical trials in human. Nevertheless, significant progress has been made in recent years to identify potent small-molecule inhibitors against the HCV protease. Clinical proof-of-concept for HCV NS3-4A protease inhibitors has recently been obtained with BILN 2061 (a non-covalent inhibitor) and VX-950 (a covalent but reversible inhibitor). Viral load in chronic hepatitis C patients was reduced by 2-3 log10 after a treatment with BILN 2061 (Lamarre et al., 2003) or VX-950 (Reesink et al., 2005) for 2–3 days. At the end of a 14-day treatment with VX-950, up to a 4-log10 reduction in HCV viral load was observed, while in some patients the virus became undetectable (<10 IU/mL) by day 14 (Reesink et al., 2005).

BIOLOGICAL FUNCTIONS

THE PRESENCE OF A SERINE PROTEASE IN HCV NS3 PROTEINIn 1989, two groups presented seminal comparative sequence studies suggesting the presence of a trypsin/chymotrypsin-like serine protease in the N-terminal one-third of the NS3 protein of flaviviruses and pestiviruses (Bazan and Fletterick, 1989; Gorbalenya et al., 1989a). Although the identity of the HCV proteins had not been determined yet at that time, these groups showed that HCV might encode a homologous trypsin/chymotrypsin-like serine protease as well. A catalytic triad of His1083, Asp1107, and Ser1165 (based on the polyprotein number of HCV-H strain) (Fig. 2A) was identified by sequence alignment of the HCV NS3 protein with many known viral and cellular serine proteases, which belong to the trypsin/chymotrypsin superfamily of serine proteases (Bazan and Fletterick, 1989; Bazan and Fletterick, 1990; Gorbalenya et al., 1989a; Miller and Purcell, 1990). In this chapter, a numerical system starting with the N terminus of the HCV NS3 protein itself will be used, in which the number of this catalytic triad would be His57, Asp81, and Ser139. Mutagenesis studies of the catalytic triad by several laboratories showed that the HCV NS3 serine protease is necessary for cleavage at four junctions in the HCV polyprotein, including NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B sites (Bartenschlager et al., 1993; Eckart et al., 1993; Grakoui et al., 1993a; Hijikata et al., 1993a; Manabe et al., 1994; Tomei et al., 1993). Substitution of any of the catalytic triad residues, His57, Asp81, or Ser139, abolished the cleavage at these four junctions, but have no effect on cleavage at other sites of the HCV polyprotein

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Fig. 2. Sequence alignment of the HCV NS3-4A serine protease and its substrates. The genotypes (1a, 1b, 2a, 2b, and 3a) are indicated at the left, and the strain names are shown in a bracket.

(A) HCV NS3 serine protease domain. The catalytic triad, His57, Asp81, and Ser139 are labeled with a filled triangle, the zinc-binding residues (Cys97, Cys99, Cys145, and His149) with an open triangle, the S1 pocket residues (Leu135, Phe154, and Ala157) with a filled arrow, and the in vitro resistance mutations (Arg155, Ala156, and Asp168) with an open arrow.

(B) HCV NS4A cofactor. The central region of NS4A that is required for activation of the NS3 serine protease is indicated with an open box. The key hydrophobic residues of NS4A that interacts with NS3 are highlighted with an underscore.

(C) The substrate sequences for the HCV NS3-4A serine protease. The scissile peptide bond is indicated by a solid arrow. A total of 10 amino acids are shown for both the P- and the P'-sides. Residues that are conserved among different cleavage sites are highlighted with an underscore.

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(C/E1, E1/E2, E2/p7, p7/NS2, and NS2/NS3), all of which are located upstream of the four NS3 serine protease-dependent cleavage sites (Fig. 1). Cleavage at the NS2/NS3 junction requires the presence of both NS2 and the N-terminal 180 residues of NS3, i.e., the serine protease domain, but not the catalytic triad of the serine protease per se. The NS2-3 protease will be the topic of another chapter in this book (reviewed in chapter 5).

THE MINIMAL DOMAIN OF SERINE PROTEASE: N-TERMINAL 180 AMINO ACIDSThe 631-residue HCV NS3 protein is a dual-function protein, containing the trypsin/chymotrypsin-like serine protease in the N-terminal region and a helicase in the C-terminal region (Gorbalenya and Koonin, 1993; Gorbalenya et al., 1989b). Co-transfection studies using constructs in which either the catalytic triad of the serine protease was mutated or the entire protease domain was deleted demonstrated that the NS3 serine protease domain, in the absence of its C-terminal helicase counterpart, is capable of mediating cleavage of polyprotein substrates (Bartenschlager et al., 1994; Lin et al., 1994; Tanji et al., 1994b). The minimal sequences required for a functional serine protease activity were determined by these groups to be the N-terminal 180 amino acids of the NS3 protein. Deletion of

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up to 14 residues from the N terminus of the NS3 protein is tolerated, although a further deletion of the N-terminal 22 amino acids resulted in significantly poorer processing of HCV polyprotein. On the other hand, deletions from C terminus of this minimal serine protease domain completely abolished proteolytic activity (Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al., 1994b).

THE HCV NS3 SERINE PROTEASE-MEDIATED CLEAVAGE OF HCV POLYPROTEINThe N-terminal amino acid sequences of the HCV NS3 serine protease-dependent cleavage products, including NS4A, NS4B, NS5A, and NS5B were determined by radioactive labeling of the HCV polyprotein with specific amino acids followed by N-terminal sequencing (Grakoui et al., 1993a). The nomenclature of Schechter and Berger (Schechter and Berger, 1967), which has been widely accepted for description of the proteases and the corresponding substrates, will be used for the HCV NS3-4A serine protease, its substrates and inhibitors in this review. A decapeptide substrate for the HCV NS3-4A protease, with 6 residues on the N-terminal side and 4 residues on the C-terminal side (Fig. 2C and 4A), would be described as NH2-P6-P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-OH, with the scissile bond locating between the P1 and P1' residues. The cleavage products of this substrate would be the P-side product (NH2-P6-P5-P4-P3-P2-P1-OH) and the P'-side product (NH2-P1'-P2'-P3'-P4'-OH). The pockets on the HCV NS3-4A protease in which the P1 side chain binds would be called the S1 pocket, and so forth for S2, S3, S4, S5, or S6 pockets and S1', S2', S3', or S4' pockets. Sequence alignment of these cleavage sites among various HCV isolates indicates that a consensus sequence would include the following elements: an acidic residue (Asp or Glu) at the P6 position, a thiol-terminating residue (Ser for the NS3/NS4A and Thr for the other three sites) at the P1 position, and a small side chain residue (Ala or Ser) at the P1' position (Fig. 2C).

Site-directed mutagenesis studies showed that blockage of any one of these four junctions has little or no impact on cleavage at other sites (Kolykhalov et al., 1994; Leinbach et al., 1994), which is consistent with kinetic analyses that have demonstrated a preferential, but clearly not obligatory, order of cleavage of the NS polyprotein by the NS3-4A serine protease (Bartenschlager et al., 1994; Lin et al., 1994; Tanji et al., 1994a). However, proteolysis of the NS3/NS4A junction is believed to be a co-translational, cis-cleavage event since an NS3-NS4A precursor were not detected and this cleavage was insensitive to dilution (Bartenschlager et al., 1994; Lin et al., 1994; Tanji et al., 1994a). The other three junctions can be cleaved by the NS3-4A serine protease in trans (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994; Tanji et al., 1994a; Tanji et al., 1994b). However, it is possible that all these proteolysis events occur in a localized, i.e. cis-cleavage environment, since the NS3-4A protease is expressed as part of the same polyprotein molecule as all of its substrates. In trans-cleavage reactions, processing at the NS5A/NS5B

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site occurred more rapidly than those at the NS4A/NS4B and NS4B/NS5A sites since rather stable NS4A-NS4B-NS5A processing intermediates were detected (Bartenschlager et al., 1994; Failla et al., 1995; Lin et al., 1994). Two additional cleavage sites in NS4B and NS5A have been identified (Kolykhalov et al., 1994; Markland et al., 1997) although their significance in viral replication is unclear. The first one is located near the N terminus of NS4B protein and its cleavage was observed only when the NS4A/NS4B cleavage was blocked (Kolykhalov et al., 1994). The second one, in the middle of NS5A protein, was seen in cell-free proteolysis experiments (Markland et al., 1997).

HCV NS4A PROTEIN IS A COFACTOR ESSENTIAL FOR ACTIVITY OF THE NS3 SERINE PROTEASEIt was demonstrated during these trans-cleavage and processing kinetics studies that an additional HCV-encoded protein, NS4A, is required as an activating cofactor for the optimal activity of the NS3 serine protease (Bartenschlager et al., 1994; Bartenschlager et al., 1995b; Failla et al., 1994; Lin et al., 1994; Tanji et al., 1995). In the absence of NS4A protein, only the NS5A/NS5B site, but not the other three cleavage sites (NS3/NS4A, NS4A/NS4B, and NS4B/NS5A), was partially processed by the NS3 serine protease alone. The presence of NS4A not only enables efficient processing at these three junctions and but also resulted in enhancement of the NS5A/NS5B cleavage. Deletion analysis showed that the central region (residues 21 to 34) of the NS4A (Fig. 2B), a 54-residue protein, is essential and sufficient for the cofactor function of the NS3 serine protease (Bartenschlager et al., 1995b; Failla et al., 1995; Lin et al., 1995; Satoh et al., 1995; Tanji et al., 1995). In addition, NS4A forms a non-covalent complex with the NS3 serine protease, which was stable in the presence of non-ionic detergent (Bartenschlager et al., 1995b; Failla et al., 1995; Hijikata et al., 1993a; Hijikata et al., 1993b; Lin et al., 1995; Satoh et al., 1995). Deletion studies indicate that the N-terminal 22 residues of NS3 and the above-mentioned central region of NS4A is involved in interaction between two proteins (Bartenschlager et al., 1995b; Failla et al., 1995; Koch et al., 1996; Lin et al., 1995; Satoh et al., 1995; Tanji et al., 1995). Substitutions that disrupted the interaction between NS3 and NS4A also resulted in reduction or loss of protease activity, suggesting that formation of an NS3-NS4A complex could be a pre-requisite for a functional serine protease (Butkiewicz et al., 1996; Koch et al., 1996; Lin et al., 1995; Shimizu et al., 1996; Steinkühler et al., 1996a; Tomei et al., 1996).

NS3 SERINE PROTEASES AND THEIR COFACTORS IN THE FLAVIVIRIDAE FAMILYThe activation of a virus-encoded protease by peptide(s) derived from another viral protein is not uncommon for viruses. This phenomenon seems to be a common feature of members of Flaviviridae family. As mentioned earlier, comparative sequence analysis suggests the presence of a chymotrypsin-like serine protease

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in the N-terminal one-third of NS3 protein of flaviviruses, pestiviruses, and HCV, which accounts for all three known genera of the Flaviviridae family at that time (Bazan and Fletterick, 1989; Bazan and Fletterick, 1990; Gorbalenya et al., 1989a; Miller and Purcell, 1990). A chymotrypsin-like serine protease was later identified in the newly discovered 4th genus of this family, hepatitis G virus (HGV) or GB virus (Leary et al., 1996; Scarselli et al., 1997). In each member of the Flaviviridae family that has been studied so far, a virus-encoded cofactor is needed to activate the corresponding NS3 serine protease. It is the NS4A protein, located immediately downstream of the NS3 protein, in the case of HCV, bovine viral diarrhea virus (BVDV) (Wiskerchen and Collett, 1991; Xu et al., 1997) of pestiviruses, and HGV or GB virus (Butkiewicz et al., 2000; Sbardellati et al., 2000). For flaviviruses, it is the NS2B protein, located immediately upstream of the NS3 protein, that fulfills the role of activator for the corresponding NS3 serine protease of yellow fever virus (Chambers et al., 1991) or dengue virus (Cahour et al., 1992; Falgout et al., 1993; Falgout et al., 1991). Again, a short peptide corresponding the central region of the NS4A cofactor of BVDV (Tautz et al., 2000) or GB virus (Butkiewicz et al., 2000), or a central 40-residue fragment of the dengue NS2B protein (Falgout et al., 1993) was sufficient for activation of the corresponding NS3 serine protease. It should be noted that there is little sequence homology between different genera with regard to these activators of the NS3 serine proteases. In fact, the NS3 serine proteases of different genera of the Flaviviridae family have quite distinct specificity for substrates, especially on the P1 residues: Cys or Thr for HCV (Grakoui et al., 1993a) and HGV or GB virus (Scarselli et al., 1997), Leu for pestiviruses (Xu et al., 1997), and Lys or Arg for flaviviruses (Chambers et al., 1991).

ADENOVIRUS PROTEASE HAS THREE ACTIVATING COFACTORSThe examples of virus-encoded proteases and their activating cofactors are not limited to Flaviviridae family. In fact, the very first example was discovered in human adenovirus. A cysteine protease encoded by human adenovirus (AVP) (for a review see Mangel et al., 2003) was found in virions and activated by an 11-residue peptide cofactor from the C-terminus of pVI protein (pVIc) (Mangel et al., 1993; Webster et al., 1993). In contrast to the HCV serine protease, the pVIc cofactor is covalently linked to the AVP by a disulfide bond (Ding et al., 1996). This protease requires binding of not one, but two co-factors, pVIc and adenovirus DNA for the optimal activity (Baniecki et al., 2001; Mangel et al., 1993; Webster et al., 1993). One possible explanation for the DNA cofactor is that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for promoter (McGrath et al., 2001). In addition, actin, a cellular cytoskeletal protein, was found to activate AVP as well (see below).

VIRUS-HOST INTERACTION OF HCV NS3-4A SERINE PROTEASESeveral recent studies suggest that HCV NS3-4A protease could be one of the weapons that HCV uses to breakdown the host antiviral response (reviewed in

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chapter 13). Innate immune response is trigged upon recognition by a family of toll-like receptor (TLR) of certain pathogen-associated molecular patterns. TLR-3 binds one of these patterns, dsRNA, a replication intermediate of many viruses, and initiates a massive type-I IFN-mediated antiviral response through activation of IFN regulatory factor 3 (IRF-3), a transcription activator. Recently, it was shown that binding of dsRNA to two DExD/H box RNA helicases, retinoic acid inducible gene I (RIG-I) and Helicard (Mda5) in cytoplasm induces a TLR-3-independent IFN response pathway, through activation of IRF-3 and NF-κB. The fact that the Huh7 hepatoma cell line, which is deficient in TLR-3-dependent pathway, became highly permissive for replication of HCV replicon RNA when an inactivating mutation in the RIG-I gene was selected in a subclone, Huh7.5 (Sumpter et al., 2005) suggests that both pathways play a role in anti-HCV immune responses. It was reported that expression of active HCV NS3-4A protease blocked activation of both TLR-3-dependent (Foy et al., 2003) and TLR-3-independent (Foy et al., 2005) signal transduction cascades, and therefore prevented the IFN-induced antiviral response against HCV RNA replication. In the case of TLR-3-dependent pathway, the substrate of HCV NS3-4A protease was reported to be Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF or TICAM-1) (Li et al., 2005). TRIF, a critical player in the TLR-3-dependent pathway, recruits two kinases, TBK1 and IKKε, to phosphorylate and activate IRF-3. In the case of TLR-3-independent cascade, the HCV NS3-4A protease was reported to cleave Cardif, a new CARD-domain containing adaptor protein that interacts with RIG-I and recruits IKKα, IKKβ, and IKKε kinases, resulting in activation of IRF-3 and NF-κB (Meylan et al., 2005).

Interference of host function by virus-encoded proteases is not limited to HCV. Actin, a cellular cytoskeletal protein, was found to interact and activate AVP (Brown et al., 2002). More specifically, it is an 11-residue peptide corresponding to the C terminus of actin, which is highly homologous to pVIc, was shown to bind and stimulate AVP activity (Brown and Mangel, 2004). The activation of AVP by actin has been proposed to be a mechanism for adenovirus to facilitate the cleavage of cytoskeletal proteins, preparing the infected cells for lysis and release of nascent virions (Brown and Mangel, 2004). In the presence of actin, the cellular skeletal protein, AVP is activated and then is able to mediate proteolysis of cytokeratin 18, another cytoskeletal protein and, not surprisingly, actin itself (Brown et al., 2002).

THREE DIMENSIONAL STRUCTURES

A DOUBLE β-BARREL FOLD OF THE HCV NS3-4A SERINE PROTEASEIn 1996, two groups presented seminal studies showing the X-ray structures of the HCV NS3 serine protease domain (Kim et al., 1996; Love et al., 1996). Kim et al.

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solved an X-ray structure of NS3 serine protease domain (residues 1 to 181) of the HCV H strain of genotype 1a in a non-covalent complex with a NS4A cofactor peptide (residues 21 to 39) (Kim et al., 1996). The HCV serine protease forms a double-barrel fold (Fig. 3A), which is similar to that of serine proteases from the chymotrypsin/trypsin super-family. The catalytic triad is located in a cleft between

Fig. 3. X-ray structures of the HCV NS3-4A serine protease. (A) Ribbon diagram of the NS3 protease domain in a complex with an NS4A cofactor peptide. The N-terminal sub-domain of the NS3 protease (in green), including the NS4A β-strand (in magenta), is on the left side and the C-terminal sub-domain (in green) on the right side. Shown in ball-and-stick are the catalytic triad, His57, Asp81, and Ser139, at the top, and the zinc atom (in cyan), which is tetrahedrally coordinated by three Cys residues (Cys97, Cys99, and Cys145) (in yellow spheres) and, via a water molecule (in red), His149 (not shown), at the bottom.

(B) A close-up view of the zinc-binding site. Shown in ball-and-stick presentation is the zinc atom (in cyan), which is coordinated by three Cys residues (Cys97, Cys99, and Cys145) (in yellow spheres) and, via a water molecule (W, in red sphere), His149.

(C) Stick diagram of interaction between the two N-terminal β-strands of the NS3 serine protease domain (thin bonds) and the NS4A activating cofactor (thick bonds). Several residues in the central region of NS4A cofactor, including Val23, Ile25, Ile29, and Leu31, make extensive hydrophobic interaction with many hydrophobic side chains of these two β-strands of the NS3 protease that form a "sandwich" with the NS4A β-strand. NS4A also forms numerous main-chain hydrogen bonds with these NS3 residues. (Reprinted from Cell (1996) 87: 343-355, J.L. Kim et al., Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. With permission from Elsevier.)

A colour version of this figure is printed in the colour plate section at the back of this book.

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two sub-domains (or barrels), with His57 and Asp81 in the N-terminal sub-domain and Ser139 in the C-terminal one. The C-terminal sub-domain (residues 96–180) contains the conventional six-stranded β barrel, common to most members of the chymotrypsin family, followed by a structurally conserved α helix. The N-terminal sub-domain (residues 1-93) consists of eight β-strands, including seven from the NS3 protein (Fig. 3A, colored in green) and one from the NS4A peptide (Fig. 3A, colored in magenta), and the latter one is sandwiched between two β-strands of the N-terminal sub-domain of NS3. In addition, the C-terminal sub-domain contains a tetrahedrally coordinated metal ion, presumably a zinc atom, located at one end of the β barrel, opposite to the catalytic triad.

EFFECTS OF NS4A BINDINGThe extensive interaction between NS3 and NS4A results in a tightly packed β-barrel and buries an additional 2400 Å2 of surface area of the NS3 protease (Fig. 3C). All but two of the main-chain carbonyl and amide groups of the NS4A residues 23–31 (Fig. 2B) form hydrogen bonds with NS3. Hydrophobic side chains of several conserved NS4A residues (Val23, Ile25, Ile29, and Leu31) (Fig. 2B) are buried in hydrophobic core of the N-terminal sub-domain of NS3 (Kim et al., 1996) (Fig. 3C). These observations in the X-ray structure confirm the previous deletion and mutagenesis studies, in which the central region of NS4A (residues 21–33), and

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in particular, the several conserved hydrophobic residues (Ile25, Ile/Val29), were shown to be critical for optimal binding of NS4A to the NS3 serine protease and activation of the protease activity (Butkiewicz et al., 1996; Lin et al., 1995; Shimizu et al., 1996; Tanji et al., 1995). The mechanism of activation by the NS4A cofactor on the NS3 serine protease activity were best illustrated by a direct comparison of three X-ray structures of HCV NS3 protease domain. In an X-ray structure of uncomplexed NS3 serine protease domain (residues 1 to 189) of the HCV BK strain of genotype 1b, solved in the absence of an NS4A cofactor, the N-terminal sub-domain of NS3 protease has only six β-strands and the N-terminal 28 residues of NS3 are extending away from the core of the protein as a flexible loop (Love et al., 1996). In two X-ray structures of the NS3 protease-NS4A cofactor complex of HCV H strain (Kim et al., 1996) or BK strain (Yan et al., 1998), respectively, the first 28 NS3 residues fold into an α-helix and a β-strand. This additional NS3 β-strand is spatially located next to the β-strand of NS4A, and both contribute to the formation of an eight-strand β-barrel (Fig. 3A). These observations in X-ray structures are consistent with earlier results that deletion of the N-terminal 22 residues of NS3 resulted to loss of the NS4A binding (Bartenschlager et al., 1995b; Failla et al., 1995; Koch et al., 1996; Satoh et al., 1995). The more striking effect of NS4A binding is on the orientation of the catalytic triad, which is highly conserved in the chymotrypsin serine protease family so that the Asp residue stabilizes the His residue after the imidazole ring of His deprotonate the OH group of the Ser nucleophile. In the absence of the NS4A cofactor, the conformation of the triad is significantly distorted so that the protease is not expected to have an appropriate activity, as observed in biochemistry studies. In the X-ray of NS3 serine protease domain alone, the imidazole ring of His57 is located too far away to effectively deprotonate the nucleophilic OH of Ser139 or to be stabilized by the Asp81 (Love et al., 1996). In the presence of NS4A cofactor peptide, the catalytic triad is in the characteristic position expected for a chymotrypsin-like serine protease (Kim et al., 1996; Yan et al., 1998).

Although both HCV serine protease and adenovirus cysteine protease require the binding of the corresponding activator(s) to be fully functional, the mechanisms of cofactor binding and activation are quite different. In the X-ray structure of human adenovirus-2 protease in a complex with its pVIc cofactor, the catalytic triad of AVP (Cys-His-Glu) adapted an arrangement similar to that of papain, and the pVIc cofactor extends a β-sheet, which is distant from the active site (Ding et al., 1996). Another difference is that pVIc binds to AVP by covalent disulfide bond and forms a 6th strand on the β-sheet (McGrath et al., 2003).

ZINC-BINDING SITEAlignment of amino acid sequences showed that three Cys and one His residues are well conserved in the NS3 protease domain of various HCV strains (Fig. 2A)

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and closely related GB viruses or hepatitis G viruses. Computational modeling analysis suggests that these four residues could form a zinc-binding site, which is located opposite to the catalytic triad in the NS3 serine protease model (Failla et al., 1996). This prediction was quickly confirmed by both X-ray structural and biochemical studies. In the X-ray structure of the NS3 serine protease domain, a zinc ion is tetrahedrally coordinated by four residues, Cys97, Cys99, Cys145, and through a water molecule, His149 (Fig. 3B) (Kim et al., 1996; Love et al., 1996). Substitution of any of the three Cys residues involved in zinc-binding with Ala led to significantly reduced protease activity, whereas mutation at His149 had much less effect (Hijikata et al., 1993a). This zinc-binding pocket is located in a cleft between two β-barrels and at the opposite end of the catalytic triad (Fig. 3A). The zinc atom is at least 20 Å away from the catalytic Ser139, suggesting the coordination of a zinc atom plays more a structural role rather than a catalytic function, as in case of several other viral proteases, including poliovirus and rhinovirus 2A cysteine proteases (Sommergruber et al., 1994; Voss et al., 1995; Yu and Loyd, 1992). A similar arrangement of Cys and His residues, as in Cys-X-Cys……Cys-X-His, where X is any residue, is also found in the cysteine protease 2A of the Picornavirae family. In these picornavirus 2A cysteine proteases, a tightly bound zinc atom is found to be critical for integrity and stability of the properly folded protein structure, rather than proteolysis. In many other serine proteases, such as chymotrypsin, a disulfide bond is found in a similar location, suggesting the zinc-binding pocket in the HCV NS3 serine protease plays a similar role as the disulfide bond in stabilizing the relative position of two β-barrels. Additional confirmation came from biochemical experiments, which showed that the purified, active HCV NS3 protease domain contained an equimolar amount of zinc (De Francesco et al., 1996; Stempniak et al., 1997). NS3 protein expressed in the absence of zinc in E. coli was not folded properly and therefore deficient of the protease activity (De Francesco et al., 1996; Stempniak et al., 1997).

THE SUBSTRATE BINDING GROOVEThe HCV NS3-4A serine protease retains some highly conserved features of the chymotrypsin family, such as spatial location of the catalytic triad of His57, Asp81, and Ser139, as well as the positions of backbone amides of Gly137 and Ser139, which forms the oxyanion hole (Kim et al., 1996). A twisted strand (residues Arg155-Ala156-Ala157-Val158) of the HCV protease superimposes well with the corresponding strand of residues in other serine proteases in the chymotrypsin family, which makes hydrogen bonds with the P3 carbonyl and the P1 and P3 amides of peptidomimetic inhibitors of serine proteases (Edwards and Bernstein, 1994). However, the HCV serine protease does display some significant difference from other serine proteases, such as chymotrypsin. For examples, several loops that interact with P4, P3, and P2 moieties of inhibitors and form a well-defined substrate-binding pocket in many other serine proteases are either shortened significantly or

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deleted in the HCV serine protease. Lack of side chain interaction is compensated by an extensive network of hydrogen bonds between the main chain atoms of the protease and substrate. However, the absence of these long loops results in a shallow, solvent-exposed substrate-binding groove and renders the design of small-molecule, peptidomimetic inhibitors against the HCV NS3-4A serine protease an extremely challenging task.

THE S1 POCKETSequence alignment of four cleavage sites in the HCV polyprotein indicates that there are three conserved positions in the HCV NS3-4A protease substrates: an Asp or Glu at P6, a Cys or Thr at P1, a Ser or Ala at P1' (Fig. 2C). Three of the cleavage sites, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B, have a Cys at the P1 position, while the NS3/NS4A site has a P1 Thr residue. In the X-ray structures, the S1 pocket is primarily determined by three hydrophobic residues, Leu135, Phe154, and Ala157 (Kim et al., 1996; Love et al., 1996). Phe154 is located at the bottom of the S1 pocket and clearly in position to make favorable van der Waals interactions with the P1 side chain. The small and hydrophobic nature of this S1 pocket is complementary to a relatively small and lipophilic side chain of a Cys. In addition, it is known that the sulfhydryl group of a Cys residue forms a favorable electrostatic interaction with the aromatic ring of Phe (Burley and Petsko, 1988). Proteolysis of substrates with larger P1 residues was allowed when Phe154 was substituted with a residue that has a smaller side chain, such as Thr, along with replacement of Ala157 with a Gly, which altered the specificity of the mutated NS3 protease (Failla et al., 1996; Koch and Bartenschlager, 1997).

THE S6 POCKETAll HCV NS3-4A cleavage sites contain an acid residue (usually Asp) at the P6 position (Fig. 2C). The P6 acid residue, sometimes along with an acidic residue at the P5 position, is believed to form electrostatic interactions with a cluster of positively charged residues of the NS3 protease, Arg123, Arg161 and Lys165 (Koch et al., 2001; Steinkühler et al., 2001).

ASSAYS FOR THE HCV NS3-4A SERINE PROTEASE

EVOLUTION OF IN VITRO BIOCHEMICAL ASSAYSWhile cell-based expression and proteolytic processing experiments were useful for providing the first glimpses of the activity of the HCV NS3-4A serine protease, their limitations were quickly reached given the complexity of cellular environment. Further understanding of the functions of this protease and substrate, which would enable meaningful drug discovery efforts, required establishment of efficient biochemical assays. In the early-generation cell-free trans-cleavage assays, the protease, the substrate or both were derived from cell lysates or in vitro translation, which may or may not have been coupled with in vitro transcription (Bouffard et al.,

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1995; Hahm et al., 1995; Koch et al., 1996; Lin and Rice, 1995; Suzuki et al., 1995; Tomei et al., 1996). Later, in vitro translated polyprotein substrates were used for monitoring activity of purified NS3 protease, which was generated as recombinant proteins in E. coli, baculovirus, or yeast expression systems (Butkiewicz et al., 1996; D'Souza et al., 1995; Markland et al., 1997; Steinkühler et al., 1996a). However, these assays using in vitro translated polyproteins are much less quantitative than those using synthetic peptides as substrates, which are cleaved by the HCV serine protease and the products are analyzed on high performance liquid chromatography (HPLC) (Bianchi et al., 1996; Inoue et al., 1998; Kakiuchi et al., 1995; Landro et al., 1997; Shimizu et al., 1996; Steinkühler et al., 1996a; Steinkühler et al., 1996b; Sudo et al., 1996; Urbani et al., 1997; Zhang et al., 1997). A colorimetric substrate, EDVVαAbuC-p-nitroanilide (5A-pNA), in which the P2 Cys was substituted with Abu and the whole P'-side replaced with a colorimetric leaving group, pNA, was used to increase assay convenience (Landro et al., 1997). It was observed when the scissile amide bond was replaced with an ester linkage, which allows ready trans-esterification of the scissile bond to the acyl-enzyme intermediate, these substrates displayed much improved catalytic efficiency (kcat/Km), allowing detection of activity with sub nM of NS3 protease (Bianchi et al., 1996). An internally quenched fluorogenic donor/acceptor couple, based on resonance energy transfer, was then incorporated into a depsipepetide substrate, which was shown to be suitable for high-throughput screening (Taliani et al., 1996).

ROLE OF NS4A COFACTORIt was shown in these studies that a synthetic peptide corresponding to the central region (residues 21–34) of NS4A could be used to substitute for the full-length NS4A protein in activation of the HCV NS3 serine protease domain (Butkiewicz et al., 1996; Lin et al., 1995; Shimizu et al., 1996; Steinkühler et al., 1996a; Tomei et al., 1996). Substitution study of NS4A peptide indicates that the following residues of NS4A, Val23, Gly27, Arg28, and in particular, Ile25 or Ile29 are critical for its cofactor function (Butkiewicz et al., 1996; Koch et al., 1996; Lin et al., 1995; Shimizu et al., 1996; Tomei et al., 1996). The activator function of NS4A peptide was largely due to an increase in the turnover rate (kcat) with a small or little change in substrate affinity (Km) for substrates corresponding to all three cleavage sites that can be cleaved in trans, namely, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B (Landro et al., 1997; Shimizu et al., 1996; Steinkühler et al., 1996a; Steinkühler et al., 1996b). The increase is more dramatic (>100-fold) for the less efficient substrate, the NS4B/NS5A substrate (Steinkühler et al., 1996b). An 1:1 stoichiometric amount of NS3 protease domain protein and NS4A peptide is sufficient for the maximal activation, suggesting the active form of HCV serine protease is a heterodimer (Steinkühler et al., 1996b). The dissociation constant (kd) was determined to be in low µM range, but the association rate (kon) was very low, suggesting conformational transitions to be the rate limiting event for the formation of NS3-4A protease complex (Bianchi et al., 1997).

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SUBSTRATE SPECIFICITYIn enzymatic assays, a decapeptide substrate, with 6 residues on the P-side and 4 more on the P'-side, was found to be optimal for efficient proteolysis by the HCV NS3-4A protease. Further truncation from either the P-side or the P'-side resulted in a significantly drop in catalytic efficiency (Landro et al., 1997; Steinkühler et al., 1996a; Zhang et al., 1997). Sequence alignment of natural decapeptide substrates for the HCV NS3-4A serine protease revealed a conserved acidic residue (Asp or Glu) at the P6, a Cys or Thr at the P1, and a Ser or Ala at the P1', resulting in the consensus substrate sequence of (Asp/Glu)–X–X–X–X–(Cys/Thr)↓(Ser/Ala)–X–X–X, whereas X indicates an variable residue (Grakoui et al., 1993a). In cell culture transfection experiments, it was found that the P6 acidic residue was dispensable and substitutions at the P1' were reasonably tolerated. However, most mutations introduced at the P1 inevitably resulted in a significant loss of proteolytic processing, indicating that the P1 Cys or, to a less extent, Thr, is the major determining factor for substrate recognition (Bartenschlager et al., 1995a; Kolykhalov et al., 1994; Komoda et al., 1994; Leinbach et al., 1994; Tanji et al., 1994a). The preference for the peptide substrate sequence in enzyme assay is also reminiscent of the consensus sequences of the HCV natural polyprotein substrates. The optimal peptide substrate in enzyme assay has an acidic residue (Glu or Asp) at P6, a Cys at P1, and a Ser or Ala at P1' (Landro et al., 1997; Urbani et al., 1997; Zhang et al., 1997). The other residues besides these three key positions (P6, P1, and P1') may also play roles in recognition by the NS3-4A serine protease, as evidenced by the drastically different catalytic efficiency in the following order: NS5A/NS5B > NS4A/NS4B >> NS4B/NS5A (Landro et al., 1997; Steinkühler et al., 1996b). These differences in catalytic efficiency are consistent with the processing order and polyprotein intermediates observed in cell culture.

CROSS TALK BETWEEN THE PROTEASE AND HELICASE DOMAINS OF NS3?In Flaviviridae family, the NS3 protein is invariably an at least bi-functional protein, with the serine protease in the N terminal one-third and the helicase in the C-terminal two-thirds. It is not unreasonable to suggest that there may be cross communication between these two enzyme functions residing in the same protein. Indeed, polynucleotides, especially poly(U), which are stimulants for the ATPase activity of the NS3 helicase, also enhanced the serine protease activity in a full-length NS3-4A protein complex purified from over-expressing COS cells (Morgenstern et al., 1997). Because the poly(U) did not stimulate the protease activity of the purified NS3 serine protease domain, these results suggest that poly(U) enhances the protease activity of the full-length NS3-4A complex through its interaction with the helicase domain, and the latter then interacts and stimulates the protease domain. The presence of the serine protease domain seems to be required for optimal binding of poly (U) to the helicase domain because the dissociation constant, Kd, of poly(U) was 10-fold lower against full-length NS3 protein than that against the helicase domain alone

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(Kanai et al., 1995). However, another study showed that poly (U) inhibited the protease activity of both protease domain alone and full-length NS3, in the presence of a synthetic NS4A cofactor peptide (Gallinari et al., 1998).

BIOCHEMISTRY OF ADENOVIRUS PROTEASE, AVPThe pVIc peptide also forms a 1:1 complex with the adenovirus protease, AVP, and enhances its catalytic efficiency by hundreds fold, while the addition of adenovirus DNA increased the kcat/Km even further. Both cofactors enhance AVP activity by increasing kcat, not by decreasing Km (Baniecki et al., 2001; Mangel et al., 1996; McGrath et al., 2001). These two cofactors bind to the AVP with a dissociation constant (Kd) in the µM range, 4.4 µM for pVIc and 0.09 µM for a 12-mer ssDNA (Baniecki et al., 2001). On the other hand, actin binds to the adenovirus protease AVP with a much lower equilibrium dissociation constant, 4.2 nM, than those exhibited by two viral, nuclear cofactors for AVP, the 11-amino acid peptide pVIc and the viral DNA (Brown and Mangel, 2004). The catalytic efficiency, kcat/Km, for substrate hydrolysis by AVP increased 150,000-fold in the presence of actin (Brown and Mangel, 2004).

A DRUG DISCOVERY TARGETEver since its identification in 1993, the HCV NS3-4A serine protease has been subjected to intense efforts on the discovery of potent, selective inhibitors as potential new therapies for the hepatitis C patients. As described above, numerous milestones on essential tools for drug discovery efforts, including biochemical and cellular assays, determination of X-ray structures, and animal models, have been achieved by many laboratories around the world during this campaign. The success of HIV protease inhibitor drugs demonstrates that viral proteases, such as the HCV NS3-4A serine protease, could be excellent targets for a structure-based drug design approach. Indeed, rational drug design has been used successfully for discovery of potent, selective inhibitors of other viral proteases, such as cytomegalovirus (CMV) proteases or rhinovirus proteases. However, in the case of HCV NS3-4A serine protease, design efforts for a small-molecule, orally available, potent, and selective drug candidate were partially hampered by the shallow, remarkably hydrophobic, substrate-binding groove of the HCV protease. Nevertheless, significant progress has been made in recent years to identify potent small-molecule inhibitors against the HCV protease. The ensuing discussion will focus on discovery of active site, peptidomimetic inhibitors, while other types of inhibitors will be briefly described.

NON-COVALENT, PRODUCT-BASED INHIBITORSIn 1998, two groups presented critical findings that, after an oligopeptide corresponding to the NS4A/NS4B or NS5A/NS5B substrate was cleaved by the HCV NS3 protease in the presence of the NS4A cofactor, the C-terminal cleavage

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product quickly dissociated from the enzyme but the N-terminal product was released rather slowly. This resulted in feedback inhibition by the N-terminal cleavage product, i.e., the hexa-peptide from the C terminus of the NS4A or NS5A, respectively (Llinas-Brunet et al., 1998b; Steinkühler et al., 1998). In addition, the free carboxylic group of the P1 residue, which is liberated by the cleavage of the substrate peptide bond, was recognized as an essential feature imparting selectivity with respect to other serine proteases (Llinas-Brunet et al., 1998a). The X-ray structure of a full-length NS3-4A protein provided an excellent explanation for the observed product-based inhibition (Yao et al., 1999). In the X-ray structure, the last residue of the NS3 protease, Thr631, which is the P1 residue of the NS3/NS4A cis-cleavage site, was bound in the active site of the NS3 serine protease domain. Apparently, after the NS3 serine protease cleaves the NS3/NS4A peptide bond, the N-terminal cleavage product, with the Thr631 as the C-terminal residue, is not released from the NS3 protease in the X-ray structure. With this seminal finding, two groups presented extensive structure-and-activity relationship (SAR) results using the hexa-peptide from the C terminus of the NS4A or NS5A, respectively. These SAR studies demonstrated that a combination of a thiol-containing residue at the P1 position and acidic residues at the P5–P6 positions is required for optimal binding and resulted in potent hexa-peptide inhibitors (Ingallinella et al., 1998; Llinas-Brunet et al., 2000). The preference displayed by NS3 protease for a thiol-containing residue, such as Cys, as the P1 anchor results from the shape of the S1 pocket. This small, hydrophilic pocket, lined by the hydrophobic residues of Val132, Leu135, and Phe154, is complementary to the small and hydrophilic side chain of Cys (Kim et al., 1996). In addition, the sulfhydryl (SH) group of the P1 Cys can interact in a unique way with the aromatic ring of Phe154. The second anchor of the hexa-peptide inhibitors is the pair of P5–P6 acidic residues, which is thought to form electrostatic interactions with a cluster of basic amino acids of the NS3 protease, including Arg123, Arg161, and Lys165 (Di Marco et al., 2000; Koch et al., 2001).

The presence of a thiol-containing side chain in the P1 position presents a major hurdle for the chemical synthesis and stability of potential inhibitors, and therefore, limits its use in clinical development (Emery et al., 1992). Substitution of P1 Cys with amino acids containing small hydrophobic side chains, such as Ala or α-aminobutyric acid, resulted in a decline in inhibitory potency due to the sub-optimal filling of the P1 subsite. Replacement with amino acids containing larger hydrophobic side chains led to a significant loss of activity presumably due to steric hindrance (reviewed in Steinkühler et al., 2001). It was shown that a difluoromethyl group is an effective mimetic of the thiol, likely due to the similarity of their steric and electrostatic properties (Narjes et al., 2002a). In addition, 1-aminocyclopropylcarboxylic acid was shown to be an effective surrogate as well, with little impact on potency when it replaced the natural P1 Cys in a hexa-peptide inhibitors (Llinas-Brunet et al., 2000).

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While the P5–P6 acidic residue pair contributes significantly to the binding of hexa-peptide inhibitors to the NS3-4A protease, it does bring two major disadvantages that render the hexa-peptide inhibitors not suitable for clinical development as oral drugs: negative charges and a rather large molecular weight (over 1,000 Daltons). The negative charges of the P5–P6 acidic pair would most likely prevent the hexa-peptides from penetrating into cells, which is reflected in the lack of cellular potency of these inhibitors in HCV replicon cells. Removal of the P5 and P6 acidic residues resulted in, as expected, a significant loss in potency of tetra-peptide inhibitors, which has to be compensated by improvement in other subsites of the inhibitors. Significant enhancement in potency was achieved with the addition of large, hydrophobic aromatic rings to the P2 Pro group, resulting in potent tetra-peptide inhibitors (Goudreau et al., 2004b). In addition, a macrocyclic ring was designed to link the side chain of the P1 and P3 residues to reduce the peptidic nature and provide rigidity to pre-order the binding conformation (Tsantrizos et al., 2003). The rigidity imparted by the ring structure constricts the molecule into exclusively adopting the correct rotamer for binding to the backbone of the NS3 protease. A 15-membered ring macrocycle was found to be optimal (Goudreau et al., 2004a). All these efforts, coupled with the previously described aminocyclopropane carboxylic acid at P1 (Rancourt et al., 2004), resulted in identification of a clinical candidate, BILN 2061 (ciluprevir) (Fig. 4A) (Llinas-Brunet et al., 2004; Tsantrizos, 2004). This compound has an excellent potency against the HCV NS3-4A serine protease, with estimated Ki values of 0.66 nM and 0.30 nM against the genotype 1a and 1b HCV protease, respectively (Lamarre et al., 2003). Treatment of the genotype 1a and 1b HCV replicon cells with BILN 2061 for 3 days resulted in a dose-dependent decrease of HCV RNA with a mean IC50 of 4 nM and 3 nM, respectively (Lamarre et al., 2003).

REVERSIBLE, COVALENT INHIBITORSThe prospect of the hexapeptide inhibitors of the HCV NS3-4A protease being developed into potential oral therapies is rather low because of their large molecular weight and the presence of multiple carboxylic acids, both of which are major obstacles for achieving high oral bioavailability. Another approach to compensate for the loss of P5–P6 acidic pair is to design a "warhead" that forms covalent bonds to the catalytic Ser nucleophile, or so-called serine-trap. Ideally, these serine-trap inhibitors will form a covalent bond to the catalytic Ser139, but this covalent bond will be cleaved so that the inhibition is not irreversible against the proteases. In the early stages of research on covalent HCV protease inhibitors, most of the standard serine-trap warheads, such as α-haloketones or heterocyclic ketones, displayed poor inhibition of this enzyme (Perni et al., 2004b). Although aldehydes were useful tools for SAR (Perni et al., 2003a), the inherent instability of aldehydes, in particular, aliphatic aldehydes, which are oxidized, rendered this warhead unsuitable for further development. The sulfonamido group is also an attractive and effective

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A

B C

Fig. 4. Chemical diagrams of the HCV NS3-4A protease inhibitors. (A) Chemical structures of the natural HCV NS5A/NS5B deca-peptide substrates (from P6 to P4'), and two NS3-4A protease inhibitors, BILN 2061 and VX-950.

(B) Schematic and (C) X-ray structure of the α-ketoamide motif bound to the NS3-4A protease active site. The inhibitor is shown at the left and the protease at the right. Two hydrogen bonds in the oxyanion hole were indicated with dashed lines. In the X-ray structure, nitrogens are shown in blue, oxygens in red, and hydrogen in white. The resolution of this X-ray structure is 2.9 Å.

A colour version of this fi gure is printed in the colour plate section at the back of this book.

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warhead for peptidomimetic scaffolds (Johansson et al., 2003). One of boronate esters demonstrated very strong, but presumably reversible, binding to the enzyme (Ki = 8 nM) (Priestley et al., 2002).

Another potent warhead is the α-ketoacid, which is capable of both covalent attachment to the catalytic Ser139 and electrostatic attraction of the carbonyl terminus (Colarusso et al., 2002). The most potent inhibitors in a dipeptide series with the α-ketoacid warhead inhibited the HCV serine protease with an IC50 of 3 µM in enzyme assay (Nizi et al., 2002). Diketones and α-ketoamides are also covalent but reversible functionalities that could serve as stable, reversible and effective binding groups (Han et al., 2003; Perni et al., 2004b), although in general, diketones are less potent than the corresponding α-ketoamides (Han et al., 2000). The covalent attachment of α-ketoamide group with Ser139, as well as an unusual interaction between the dicarbonyl motif and the oxyanion of the protease (Fig. 4B), provide exceptional potency against the HCV NS3-4A protease. This class of inhibitors has a slow-binding mechanism of action due to the requirement of an unusual re-arrangement in the active site of the NS3 similar to that observed for ketoacids (Liu et al., 2004; Perni et al., 2004b). Extensive optimization of the P3 and P4 hydrophobic groups, removal of the acidic charge at P1' and, finally, the modification of the P2 Pro substituent into a bicyclic Pro motif (Perni et al., 2003a; Perni et al., 2004a; Perni et al., 2004b; Sun et al., 2004; Victor et al., 2004; Yip et al., 2004a; Yip et al., 2004b) resulted in identification of a clinical candidate, VX-950 (Fig. 4A) (Perni et al., 2003b). While the optimal length for recognition by the HCV NS3-4A protease is 10 amino acids in natural HCV substrates, the backbone of these inhibitors was truncated to a tetrapeptide scaffold while maintaining significant binding affinity for the NS3-4A serine protease (Perni et al., 2003b). This compound had excellent potency against the HCV NS3-4A serine protease, with a Ki* value of 7 nM against the genotype 1a HCV protease (Perni et al., 2003b). Despite of the large difference in the IC50 values in a 2-day replicon cell assay, these two inhibitors had a comparable ability to induce a 4-log10 reduction of HCV RNA levels after an extended incubation with replicon cells (Lin et al., 2003; Lin et al., 2006).

NON-COVALENT SUBSTRATE MIMETICS AND P' INHIBITORSTo date, most peptidomimetic, active site inhibitors of the HCV serine protease were designed against those binding pockets on the P-side, and much less effort has been spent on the P'-side. It was found that the replacement of the P1' residue of a decapeptide substrate based on the NS5A/NS5B site led to poor turnover of the substrate by the HCV protease (Steinkühler et al., 1996b; Urbani et al., 1997), indicating that the S1' pocket can accommodate residues much larger than the natural Ser residue. In addition, a favorable interaction was found between the P4' residue (Tyr) of these inhibitors and the NS4A cofactor (Landro et al., 1997).

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Incorporation of the optimized P'-side sequence and an N-terminal carboxylic acid, which is well-positioned in the active site to engage in interactions similar to those previously described for the C-terminal carboxylic acid of non-covalent product-based inhibitors, resulted in a novel series of HCV NS3-4A protease inhibitors that bind exclusively to the P'-side, without any contact with the P-side of the enzyme (Ingallinella et al., 2002). Competitive, capped tripeptide inhibitors of the NS3-4A protease, with low µM potency, were identified with the addition of a proper linkage between these two elements from a small combinational library. Another series of reversible, competitive inhibitors binding to the substrate-binding cleft across the active site has been described (Colarusso et al., 2003). The presence of a C-terminal phenethylamide group in these inhibitors allows an interaction with the S'-side of the enzyme, which might present a potential alternative to the C-terminal free carboxylic group present in the non-covalent, product analogue inhibitors.

IRREVERSIBLE INHIBITORSAs described above, the vast majority of HCV NS3-4A protease inhibitors that have been described to date are either non-covalent or covalent but reversible inhibitors. Recently, a series based on a pyrrolidine-5,5-trans-lactam core was reported to inhibit the HCV protease with an IC50 up to 0.30 µM in replicon cell assay. These inhibitors bind irreversibly to the enzyme through opening of the lactam ring, with a biochemical potency (kobs/I) of 7,760 M-1s-1 for the best compound in this series (Andrews et al., 2003a; Andrews et al., 2002; Andrews et al., 2003b; Andrews et al., 2003c; Slater et al., 2002).

INTERACTION BETWEEN NS3 AND NS4ABecause the interaction with the NS4A co-factor is critical for maintenance of a stable, active conformation of the NS3 protease, it is thought that agents competing against the NS4A binding could be used to inhibit the HCV protease activity. A 14-mer NS4A peptide (residues 21–34) with a substitution of Arg28 with Glu yielded an IC50 of 20 µM against activation of the NS3 protease by the wild-type NS4A peptide (Shimizu et al., 1996). 13-mer NS4A analogs (residues 22–34) assembled from D-amino acids (instead of the normal L-amino acids) in a standard order, or from L-amino acids in a reverse order, inhibited the NS3 protease activity with an IC50 of 0.2 µM (Butkiewicz et al., 1996; Walker, 1999). In addition, several bivalent inhibitors in which the above-mentioned NS4A analogs were fused in frame to a NS5A/NS5B substrate have been described with IC50 values ranging from 0.2 µM to 3 µM. In these cases, a hexapeptide (Glu-Asp-Val-Val-Cys-Cys), corresponding to a P6–P1 portion of the NS5A/NS5B substrate, was fused via a short linker of 1–3 amino acids to the NS4A analogs (Walker, 1999). However, it is unclear how these NS4A analogs inhibit the NS3-4A protease activity. Furthermore, since formation of the NS3-NS4A non-covalent complex may occur co-translationally during synthesis of the HCV polyprotein, it remains to be seen whether these NS4A

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analogs will be able to effectively compete against an already formed, tightly bound NS3-NS4A complex.

APTAMERSAnother strategy to inhibit the HCV NS3-4A protease is to select aptamers (Biroccio et al., 2002; Fukuda et al., 1997; Fukuda et al., 2000; Sekiya et al., 2003; Urvil et al., 1997), which are single-stranded nucleic acids binding into a specific pocket of the target protein with high affinity and interfering with function(s) of the protein. Aptamers can be identified via multiple rounds of selection and amplification from a pool of random nucleic acids against any protein or small-molecule target. Two of these aptamers inhibit the HCV NS3 serine protease with an excellent potency (Ki = 3 µM for the better aptamer inhibitor) (Kumar et al., 1997). In addition, the same two aptamers were shown to block the helicase activity of NS3 (Kumar et al., 1997). Inhibition of the NS3 protease activity by a different set of RNA aptamers (Fukuda et al., 2000; Sekiya et al., 2003) was also demonstrated in transfected cells (Nishikawa et al., 2003).

OTHER NON-PEPTIDIC SMALL-MOLECULE INHIBITORSSeveral groups have undertaken high-throughput screening of large libraries of chemical or natural products to identify novel inhibitors of the HCV NS3-4A serine protease that are not peptidomimetic. Many of the confirmed hits are non-competitive against the substrates (for a detailed review see Beaulieu and Llinas-Brunet, 2002). One of the hit series is 2,4,6-trihydroxyl-3-nitrobenzamides (THNBs) with an IC50 of 3.0 or 5.8 µM in the absence or presence of NS4A, respectively (Sudo et al., 1997b). However, the major challenge for THNBs as potential therapeutic agents against HCV is the lack of selectivity against human serine proteases, such as chymotrypsin and elastase. Another series with a thiazolidine core was identified by the same group of scientists, with an IC50 of 2.3 µg/mL (Sudo et al., 1997a). Again, improvement in selectivity is the major challenge for this series as the most selective derivative in this series showed a slight decline in potency. In a recent structure-based NMR screening of a customized fragment library, 16 small-molecule hits were discovered to bind weakly, with a Kd in the range of 100 µM to 10 mM, to substrate binding sites of the HCV NS3-4A protease (Wyss et al., 2004). NMR chemical shift perturbation data were then used to identify the binding location and the orientation of the active site directed scaffolds. Two of these compounds, which bind at the proximal S1–S3 and S2' substrate binding pockets, were linked together to generate competitive inhibitors with relatively high molecular weight and IC50 values in the µM range (Wyss et al., 2004). Novel, Zn2+-dependent benzimidazole-based inhibitors of the HCV serine protease have also been reported to form a ternary complex with a Zn2+ ion and the catalytic residues, His57 and Ser195, as determined in X-ray crystallography. However, the SAR for this series of compounds was established using a Zn2+-independent system, and did not appear to be consistent in the presence of Zn2+ (Sperandio et al., 2002).

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The highly charged nature and the dependence on a high concentration of Zn2+ ion of this class of inhibitors present significant challenges in terms of cellular penetration and oral bioavailability. Finally, a β-sheet mimetic has been combined with the boronate ester warhead to create a potent series of inhibitors (Glunz et al., 2003; Zhang et al., 2003).

PRECLINICAL STUDIES AND CLINICAL DEVELOPMENT

IN VITRO RESISTANCE MUTATIONSBecause of the error-prone nature of the viral reverse transcriptase of retroviruses or RNA-dependent RNA polymerase of RNA viruses, drug resistance frequently emerges in patients treated with antiviral drugs and therefore limits the efficacy of these therapies. For new HCV NS3-4A serine protease inhibitors, resistance could become a major issue in the treatment of patients. The HCV subgenomic replicon system (Blight et al., 2000; Lohmann et al., 1999) was used for identification of in vitro resistance mutations against two HCV protease inhibitor clinical candidates, BILN 2061 or VX-950. All of the in vitro resistance mutations selected against either inhibitor were substitutions of a single amino acid in the NS3 serine protease domain and resulted in significant reduction in susceptibility to the respective inhibitor (Lin et al., 2005a; Lin et al., 2004a; Lu et al., 2004). Two of the primary resistance mutants against BILN 2061, Asp168-to-Val (D168V) and Asp168-to-Ala (D168A), were highly resistant to BILN 2061 as reflected in at least a 63-fold increase in Ki values in the FRET substrate-based enzyme assay and a more than several hundred-fold jump in the IC50 values in the subgenomic replicon cell assay (Lin et al., 2004a). However, both mutants at Asp168 remained fully susceptible to VX-950 because there was only a slight decrease in both Ki and IC50 values in enzyme and replicon cell assays, respectively (Lin et al., 2004a). Asp168 is in salt-bridge interactions with the side-chains of Arg123 and Arg155, and is also part of the S4 binding pocket. Computational modeling analysis suggests that substitution of Asp168 with a non-acidic residue, such as Val or Ala, results in the loss of salt-bridge interaction with the Arg155 side-chain on the neighboring β-strand (Fig. 5, color coded in light green), which in turn makes multiple contacts with the large P2 group of BILN 2061 in the model. Therefore, the conformation of the Arg155 in the BILN 2061-wild-type NS3 protease complex is no longer energetically favored in the D168V or D168A mutant. Instead, Arg155 in these two mutants appears to clash with the P2 quinoline group of BILN 2061 and destabilizes its binding. On the other hand, the conformation of Arg155 in the two published crystal structures of the NS3 protease-inhibitor complex is similar to that in the VX-950-protease complex (Fig. 5, color coded in orange). In addition, this conformation of Arg155 confers stabilization of VX-950 binding as it allows the maximal number of van der Waals contacts between the Arg155 side-chain and the inhibitor. Therefore, VX-950 is not expected to be affected by the substitutions at Asp168 compared with

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BILN 2061 (Lin et al., 2004a). It should be noted that substitutions at Asp168 have been identified in a previous study as the resistance mutations against a less potent HCV protease inhibitor, which had an IC50 of about 1 µM in the replicon cell assay (Trozzi et al., 2003). Another BILN 2061-resistant mutation, substitution of Arg155 with Gln (R155Q), was identified in a separate in vitro study. The R155Q mutant was moderately resistant to BILN 2061 (a 24-fold increase in replicon cell IC50) (Lu et al., 2004), although it is not clear whether this mutation confers resistance to VX-950 or not.

The major in vitro resistance mutant against VX-950, Ala156-to-Ser (A156S), was moderately resistant to VX-950 with a ~12-fold and ~29-fold increase in enzyme Ki and replicon cellular IC50 values, respectively (Lin et al., 2004a). The HCV

Fig. 5. Computational models of the HCV NS3-4A protease in complex with its inhibitors. The protein is shown as a schematic based on its secondary structure in light gray. The inhibitors (VX-950 in yellow and BILN 2061 in cyan) are shown as ball-and-stick models, with nitrogens in blue, oxygens in red, and sulfur in orange. The side chains of Ala156 (green), Asp168 (orange) and Arg123 (orange) are shown as sticks. The Arg155 side-chain of BILN 2061-protease model (R155-BI) is shown in light green and that of VX-950-protease model (R155-VX) in orange. The catalytic triad, Ser139, His57, and Asp81 is shown in gray. The figure was created using PyMOL Molecular Graphics Systems (DeLano Scientific LLC, San Carlos, California).A colour version of this figure is printed in the colour plate section at the back of this book.

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replicon cells containing the A156S substitution remained as sensitive to BILN 2061 as the wild-type replicon cells (Lin et al., 2004a). The Ala156 side-chain is in van der Waals contact with the P2 group of these two inhibitors (Fig. 5, color coded in green). In a computational model of the A156S mutant, the terminal oxygen of Ser156 is too close to the P4 cyclohexyl group of VX-950, and it is also close to the terminal cyclopentyl cap of BILN 2061. Because the cyclopentyl cap of BILN 2061 is at the flexible end of the inhibitor, it can be moved away from this unfavorable contact without significantly affecting BILN 2061 binding. A similar movement of the P4 cyclohexyl group of VX-950 causes destabilization of the interactions between the inhibitor and S4 and S5 sub-sites of the protease. Therefore, a larger loss in binding affinity is expected for VX-950 than for BILN 2061 with the A156S mutant protease.

The lack of overlap between the dominant in vitro resistance mutations of BILN 2061 and VX-950 raised an interesting question – whether it is possible for a combination of these two protease inhibitors to suppress the emergence of these resistance mutations. While the combination did suppress the occurrence of A156S, D168V or D168A mutants, two other single-residue substitutions, Ala156-to-Thr (A156T) and Ala156-to-Val (A156V), were selected and found to confer cross-resistance to both VX-950 and BILN 2061 (Lin et al., 2005a). In a computational model, two out of the three possible conformations of the A156S side chain have unfavorable contacts with both the inhibitors either at the P2 side chain or P3 carbonyl group. In the A156T or A156V mutation, the additional hydroxyl or methyl group, respectively, at the Cβ atom of the residue 156 side-chain is forced to occupy one of these two unfavorable positions, which leads to a repulsive interaction with the inhibitor and/or enzyme backbone atoms. Therefore, A156T and A156V mutants are expected to be resistant to both inhibitors (Lin et al., 2005a). It also remains to be seen which of these resistance mutations identified in cell culture, if any, will be observed in patients treated with HCV protease inhibitors.

IN VITRO COMBINATIONSThe current standard care for hepatitis C is a combination of weekly injections of pegylated IFN-α and daily oral doses of ribavirin, which results in a SVR in roughly half of treated patients. The SVR is higher (~80%) in patients infected with genotype 2 or 3 HCV, but much lower (40-50%) in genotype 1 HCV-infected patients, who account for the majority of hepatitis C population in developed countries. It remains to be seen whether a single direct antiviral agent, such as a protease inhibitor, is sufficient to induce a more favorable SVR in chronically infected hepatitis C patients. One possible strategy to increase efficacy and help suppress the emergence of resistance mutations in HCV protease inhibitor-based therapy is to combine it with other antiviral agents, such as IFN-α or a polymerase inhibitor. It has been shown that a combination of an HCV NS3-4A protease inhibitor

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with IFN-α resulted in a synergistic reduction of HCV RNA in the subgenomic replicon cells after a 2-day incubation (Lin et al., 2004b). Furthermore, the benefit of the combination was sustained over time such that a nearly 4-log10 or 10,000-fold reduction of HCV RNA was achieved following a 9-day treatment of the HCV replicon cells. The viral RNA dropped by more than 4-log10 to below the detection limit after a-14 day combination treatment.

In the presence of G418 (neomycin), which allows selective growth of replication-competent HCV replicon cells over "cured" Huh7 cells, no replicon cells were recovered three weeks after withdrawal of the inhibitors, suggesting that the HCV RNA has been cleared from the cells by the 14-day combination treatment (Lin et al., 2004b). In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which potentially may reduce the risk of possible adverse effects associated with high doses of either agent (Lin et al., 2004b). Given the observation that HCV protease may interfere with the IFN signal transduction pathway, one of the major components of the host anti-viral response, these data suggest that HCV protease inhibitors may have a dual anti-HCV function, blocking the HCV RNA replication and restoring the host antiviral response.

CLINICAL DEVELOPMENTBILN 2061 (Fig. 4A, ciluprevir) was the first NS3-4A protease inhibitor to enter clinical development. Despite its peptidic nature, BILN 2061 showed a low-to-moderate oral bioavailability after a single dose in multiple species of animals, including rats and dogs (Lamarre et al., 2003; Narjes et al., 2002b). In a single-dose escalation phase 1a trial in healthy adults, this compound showed dose-proportionality up to the 1,200 mg dose, and was well tolerated up to the 2,000 mg dose. In several two-day, twice-daily dosing studies, BILN 2061 has been shown to possess proof-of-concept antiviral activity in chronic genotype 1 HCV-infected patients with minimal or advanced fibrosis (Benhamou et al., 2002; Hinrichsen et al., 2002; Lamarre et al., 2003). At 200 mg/administration, a 2-3 log10 or greater reduction in viral load was observed after the 2-day treatment. In some patients, the viral load dropped below the limit of detection using a relatively insensitive assay (<1500 copies/mL), although it remained positive in a more-sensitive assay (>50 copies/mL). However, BILN 2061 was much less effective against genotypes 2 and 3 HCV, as evidenced by the Ki values (80–90 nM) against these proteases in vitro (Thibeault et al., 2004), and an uneven, less pronounced viral load reduction in the genotypes 2 and 3 HCV infected patients (Reiser et al., 2003). Unfortunately, further development of BILN 2061 was put on hold due to safety concerns in animals, which has not yet been fully disclosed.

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As described before, VX-950 (Fig. 4A) has a different mechanism of inhibition than BILN 2061. VX-950 was the first covalent, reversible inhibitor of the HCV NS3-4A protease to enter clinical development for hepatitis C. VX-950 showed a moderate oral bioavailability and a much higher exposure in the livers than in the plasma after a single dose in both rats and dogs (Perni et al., 2003b). In a single-dose escalation (range 25–1,250 mg) phase 1a trial in healthy adults, VX-950 was well tolerated up to the 1,250 mg dose level and exhibited good systemic exposure, which was greater than proportional to dose (Chu et al., 2004). In a recently completed 14-day study, VX-950 demonstrated excellent antiviral activity in chronic genotype 1 HCV-infected patients (Reesink et al., 2005) (Fig. 6). Again, VX-950 was well tolerated in all three groups, 450 mg thrice daily, 750 mg thrice daily, and 1,250 mg twice daily. In all three groups treated with different VX-950 regimens, a 2–3 log10 or greater reduction in viral load was observed after the first 2 days of treatment, as in the case of BILN 2061. In some patients, the viral load dropped by more than 4 log10 to below the limit of detection of a very sensitive assay (<10 IU/mL) after 14 days of dosing. In addition, VX-950 was equally potent

Fig. 6. Antiviral antivity of VX-950 in chronic HCV-infected patients. Chronic genotype 1 HCV-infected patients were treated with VX-950 at the following doses for 14 days: 450 mg thrice daily (open square, n=10), 750 mg thrice daily (filled diamond, n=8), 1250 mg twice daily (filled triangle, n=10), or placebo (open circle, n=6). The first dose was given at day 1, as indicated by a dashed line. The plasma HCV RNA level was measured using COBAS Taqman HCV RNA assay, and the mean plasma viral load of each treatment group is shown.

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against the HCV NS3-4A protease of genotype 2, but not genotype 3, in enzyme assays (Taylor et al., 2004). A close homolog of VX-950 inhibited the replication of a genotype 2a full-length HCV replicon that is capable of generating infectious virus particle (Lindenbach et al., 2005), suggesting that it may also be an effective agent for genotype 2 HCV-infected patients.

CONCLUSIONS AND FUTURE DIRECTIONSThe current standard therapy for chronic hepatitis C patients is a combination of weekly injections of pegylated IFN-α, and daily oral doses of ribavirin. Both drugs are indirect antiviral agents because they do not target a specific HCV protein or nucleic acid. A SVR is achieved in only half of the treated patients and in less than half of patients with genotype 1 HCV or with high viral load. The standard therapy is associated with considerable adverse effects. There is a large unmet medical need for orally available, small-molecule, direct anti-HCV drugs to provide hepatitis C patients more effective treatments with fewer side effects. Ever since the determination of HCV genome sequences in 1989 (Choo et al., 1989; Kuo et al., 1989) and the identification and characterization of HCV proteins in the early 1990's, there have been intense efforts to discover novel direct antiviral drugs against HCV. Determination of X-ray structures of the HCV NS3-4A serine protease and the success of HIV protease inhibitors raised the hope of using structure-based approaches to design a protease inhibitor against HCV. However, discovery of a small-molecule, orally available, and potent drug candidate have been partially hampered by the shallow substrate-binding groove of the HCV NS3-4A serine protease. In addition, the lack of a robust small animal model for HCV infection has generally forced scientists to rely on a combination of anti-HCV activity in cell culture and animal pharmacokinetics as surrogate indicators of efficacy prior to human trials. Nevertheless, significant progress has been made in recent years to identify potent small-molecule inhibitors against the HCV protease. Clinical proof-of-concept for HCV NS3-4A protease inhibitors has recently been obtained with two inhibitors, BILN 2061 and VX-950. Given the lack of proof-reading function of the HCV NS5B RNA-dependent RNA polymerase, potential drug resistance is still a major concern for any direct antivirals against HCV, as in the case of HIV. It remains to be seen whether HCV NS3-4A serine protease inhibitors will be used as a monotherapy or in combination of other drugs, such as IFN-α, a polymerase inhibitor, or both. Nevertheless, it will be very exciting to see HCV NS3-4A serine protease inhibitors progress through clinical developments and, hopefully, provide hepatitis C patients with much needed, more effective therapies.

ACKNOWLEDGMENTSThe author would like to thank Michael Briggs, Robert Kauffman, Steve Lyons, Robert Perni, and John Thomson for the critical reading and editorial comments on the manuscript.

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Chapter 7

HCV Helicase: Structure, Function, and Inhibition

David N. Frick

ABSTRACTThe C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled by ATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.

INTRODUCTIONThe C-terminal two thirds of the HCV NS3 protein forms an enzyme that is seemingly unrelated to the serine protease discussed in the previous chapter. The section of NS3 not needed for polyprotein cleavage folds into a three-domain molecule that rapidly hydrolyzes all natural nucleoside triphosphates (NTPs) and uses the resulting energy to move along a nucleic acid polymer dislodging a complementary strand or bound proteins. All cells and many viruses express similar proteins, which are called "helicases" because their biological role is normally to unwind a DNA double helix. Since there is no DNA stage in the HCV lifecycle, the exact function of HCV helicase is still unclear. It could unwind duplex RNA that is formed when the single-stranded HCV genome is copied, or it might smooth RNA secondary structures, which impede the NS5B RNA-dependent RNA polymerase. Alternatively, the ability of HCV helicase to move like a motor along RNA could be used for a process not linked to bona fide helicase activity (i.e. the disruption of base pairs). HCV helicase could strip RNA binding proteins from viral RNA, assist translation, or even help coordinate translation and polyprotein processing. Regardless of its precise role in the HCV lifecycle, HCV helicase activity seems necessary for viral replication, as evidenced by the fact that a mutated infectious

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clone, in which a point mutation abolishes the ability of NS3 to hydrolyze ATP, does not replicate in chimpanzees (Kolykhalov et al., 2000).

In the last review devoted entirely to HCV helicase, Kwong et al. discussed its basic biochemical properties, assay conditions, and the functions of conserved motifs as revealed by the first crystal structures and initial structure-based site-directed mutagenesis (Kwong et al., 2000). Several other reviews have also discussed some of that material (Korolev et al., 1998; Yao and Weber, 1998; Frick, 2003; Frick, 2004). This chapter will therefore only briefly review many of these topics, and will instead focus mainly on more recent mechanistic insights and HCV helicase inhibitors that have been reported.

STRUCTURE OF HCV HELICASEUnlike other systems where mechanistic experiments were carried out long before protein-substrate interactions were viewed at an atomic resolution, the first crystal structures of HCV helicase were solved only a few years after the protein was first purified (Yao et al., 1997; Cho et al., 1998; Kim et al., 1998; Yao et al., 1999). These structures are shown in Fig. 1. The helicase portion of NS3 forms three domains. When viewed as a Y-shaped molecule, the most N-terminal domain (domain 1) and the middle domain (domain 2) are above the C-terminal domain (domain 3). In one structure (Kim et al., 1998), a short DNA oligonucleotide, containing only the RNA base uracil, is bound to the helicase in the cleft that separates domain 3 from domains 1 and 2 (Fig. 1A). In several structures, a sulfate molecule is seen bound between domains 1 and 2, in a position where ATP has been seen in high-resolution structures of similar helicases (Soultanas et al., 1999; Velankar et al., 1999; Bernstein et al., 2003) (Fig. 1A, C). When the entire NS3-NS4A complex is viewed with the ATP and DNA binding sites in the front, the protease is in the back, with its active site buried on the back of the helicase domains (Yao et al., 1999) (Fig. 1C). Behind the protease is its NS4A cofactor, which positions the catalytic triad of the protease so it will cleave the NS3-4A junction (Kim et al., 1996). The zinc ion needed for the NS2-3 auto-catalytic protease lies on the same side of the protease as NS4A (Fig. 1C). These crystal structures, along with high resolution NMR structures of HCV helicase domain 2 (Liu et al., 2001; Liu et al., 2003), have greatly influenced proposals explaining how helicases function and have guided experiments designed to test these ideas.

Fig. 1. HCV Helicase Structures. A. PDB file 1A1V, showing the HCV helicase with a bound DNA oligonucleotide and sulfate ion (Kim et al., 1998). The N-terminal RecA-like domain (domain 1) is colored yellow, the C-terminal RecA-like domain (domain 2) is purple, and domain 3 is pink. DNA and a sulfate ion (which occupies the ATP binding site) are depicted as spheres. (B) An electrostatic surface of the protein in 1A1V calculated without the DNA using the program APBS (Baker et al., 2001). Note the DNA is held in a negatively-charged pocket. (C) A full-length NS3 complex with the central portion of NS4A covalently tethered to the NS3 N-terminus (Howe et al., 1999), as seen in

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PDB file 1CU1 (Yao et al., 1999). Helicase domains are colored as in panel A with the protease colored green and NS4A blue. The protein is rotated about 90º relative to panel A. (D) An electrostatic surface of the protein as viewed in panel C. Note that the positively-charged cleft surrounding the protease, which could provide additional RNA binding sites. (E) Comparison of HCV helicase in the closed conformation (PDB file 1HEI Yao et al., 1997) and the open conformation (PDB file 8OHM, Cho et al., 1998). Proteins are superimposed along domains 1 and 3. (F) The model for a HCV helicase dimer that was proposed by Cho et al. (1998). Protein domains are colored as in panels A and C. All structures were rendered using the program Pymol (DeLano Scientific LLC, San Francisco, CA).A colour version of this figure is printed in the colour plate section at the back of this book.

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STRUCTURAL VARIATIONSEach of the available HCV helicase crystal structures is shown in one of the panels of Fig. 1. Kim et al.'s structure (PDB accession code 1A1V, Kim et al., 1998) is shown in Fig. 1 panels A and B, and Yao et al.'s structure (PDB 1CU1, Yao et al., 1999) is in panels C and D. In panel E, Cho et al.'s structure (PDB 8OHM, Cho et al., 1998) and Yao et al.'s structure (PDB 1HEI, Yao et al., 1997) are aligned for comparison. Finally, Cho et al.'s model for a helicase dimer is shown in Fig. 1F. The main difference between the available HCV structures concerns the position of domain 2 relative to domains 1 and 3. Domains 1 and 3 share more of an interface than domain 2 shares with either of the other domains. Domain 2 is connected to domains 1 and 3 via flexible linkers, which allow domain 2 to freely rotate relative to domains 1 and 3. In some structures, domain 2 is rotated away from domain 1 in an "open" conformation, while in other structures domain 2 is closer to domain 1 in a "closed" conformation (Fig. 1E). The pivot point for these rotations is provided by additional contacts between domain 3 and an extended β-hairpin originating from domain 2. An animation showing the rotation of domain 2 is available in the Database of Macromolecular Movements (http://www.molmovdb.org/cgi-bin/morph.cgi?ID=109065-518) (Echols et al., 2003).

How well these structures represent the diverse array of NS3 proteins encoded by all varieties of HCV is not clear because natural variation in the amino acid sequence of NS3 undoubtedly impacts its structure. The known HCV genotypes have remarkably different nucleotide sequences, and the corresponding amino acid substitutions likely would affect protein folding. HCV helicase from only three, very similar, genotypes has been examined at the atomic level. Both Yao et al. (1997) and Kim et al. (1998) examined an enzyme isolated from the same genotype 1a H strain, Yao et al. (1999) examined the helicase from the genotype 1b BK strain, and Cho et al. (1998) used an enzyme from another genotype 1b strain. Although there are many differences between the structures, no obvious differences appear to be genotype specific; the genotype 1a structures are as different from each other as they are from the genotype 1b structures.

Nevertheless, variation in HCV helicase residues clearly influences its activity, as evidenced by the fact that adaptive mutations in HCV replicons (see Chapter 11) frequently arise in the helicase region (Blight et al., 2000; Krieger et al., 2001; Grobler et al., 2003). To concisely depict helicase sequence variability among various HCV genotypes, a consensus sequence of the NS3 peptide is superimposed on a cartoon of the helicase structure in Fig. 2. This figure was generated by rendering an alignment of all the NS3 sequences deposited in the hepatitis C virus (HCV) database project (http://hcv.lanl.gov/) using a program called Weblogo (http://weblogo.berkeley.edu/) (Crooks et al., 2004). The original alignment can be downloaded from the HCV database by choosing "NS3" under the subheading

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"alignments." Weblogo depicts an alignment as a sequence logo (Schneider and Stephens, 1990), in which each NS3 residue is represented as a stack of one letter amino acid codes. The height of each stack corresponds to the amino acid conservation at that position. When the residue is invariant, only one letter is shown, and the most common substitutions are noted when the residue is variable.

Lam et al. (2003b) have explored the impact of genotypic variation on the various activities of HCV helicase by examining recombinant proteins that were isolated from infectious clones of HCV genotype 1a (Yanagi et al., 1997), 1b (Yanagi et al., 1998), and 2a (Yanagi et al., 1999). Although there are some differences between the genotypes, the proteins are surprisingly similar. The main difference between genotype 1 and 2 strains can be attributed to variation at residue 450, which is normally a Thr (Fig. 2), but in the genotype 2a infectious clone it is an Ile

Fig. 2. HCV NS3 sequence conservation. A sequence logo (Schneider and Stephens, 1990) of an NS3 sequence alignment is overlaid on a cartoon of the HCV helicase. Each residue of NS3 is depicted as a stack of letters, the height of which correlates with how well it is conserved in 138 NS3 sequences deposited in the HCV database (http://hcv.lanl.gov/). The height of the letters in each stack correlates with how frequently that amino acid occurs at that position. Conserved superfamily 2 helicase motifs and other key residues are noted and highlighted with bold type.

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(Yanagi et al., 1999). When Thr450 alone is changed to Ile, the protein binds ssDNA differently and unwinds DNA faster, suggesting that the interaction of Thr450 with DNA observed in the crystal structure somehow modulates the rate of helicase movement. Upon close examination of sequences that were later deposited in the HCV database, it appears now that only the particular genotype 2a strain used in our study (Lam et al., 2003b) contains the Ile substitution. Since this was an infectious clone isolated from a chimpanzee (Yanagi et al., 1999), we now believe that T450I is an adaptive mutation that permits the virus to efficiently replicate in chimpanzees, but it is not normally seen in HCV infecting humans.

CONSERVED MOTIFSThe sequence logo in Fig. 2 also reveals that there are numerous stretches of amino acids that do not vary in known isolates. The numbered sequence motifs are shared with related helicases (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). Some of these motifs, such as the DExD/H-box portion of motif II and motif IV are characteristic only of helicases closely related to HCV helicase, while others, such as the Walker A motif (Motif I), are conserved among all helicases and in a wide variety of other proteins that hydrolyze ATP. Other motifs are found only in HCV and closely related viruses, including the Arg-clamp, the Phe loop (Lam et al., 2003a), and all motifs in domain 3.

It was not possible to precisely depict all the conserved residues in Fig. 2 relative to their position within each domain of HCV helicase, but most are close. Diagrams noting exact motif positions on actual crystal structures have been published elsewhere (Hall and Matson, 1999; Kwong et al., 2000; Lam et al., 2003a; Frick, 2004). Motifs I, Ia, II, III, IV, V, and VI, which are conserved in similar helicases encoded by both viruses and cellular organisms, line the ATP binding cleft, and some of these motifs project residues into the nucleic acid binding site. These seven helicase motifs essentially form the motor which converts the chemical energy derived from ATP hydrolysis into a mechanical force that drives helicase movements leading to the disruption of DNA or RNA base pairs.

The roles of most of the key conserved residues in motifs I through VI have been investigated using site-directed mutagenesis. The various studies are tabulated in Table 1 along with a phenotype of each mutant. The phenotype listed in Table 1 simply depicts whether helicase activity (i.e. DNA or RNA unwinding), ATPase, or nucleic acid binding properties of each mutant is unchanged (normal), diminished, or enhanced. Mutations in motifs I-VI normally impact the ability of the protein to both unwind DNA and hydrolyze ATP, showing that the two activities are coupled. However, sometimes mutations result in decreases of ATP hydrolysis rate, but not a similar corresponding decrease in DNA unwinding. The results have been interpreted by some authors as evidence that ATP hydrolysis is not absolutely

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required for unwinding, but they could instead be due to the different sensitivities of ATPase and helicase assays under the particular conditions utilized.

Outside motifs I to VI, domains 1 and 2 have regions that are both variable and conserved. With a hope of discovering regions that might provide binding sites for novel anti-HCV therapeutics, our lab examined the role of two motifs in domain 2 that are conserved in all HCV isolates but not related proteins. The rationale was that compounds that bind such sites would be relatively non-toxic because similar sites are not present on related cellular helicases. The first motif identified centered on Arg393, a residue that contacts the nucleic acid backbone. When Arg393 is changed to Ala, the protein still catalyzes RNA-stimulated ATP hydrolysis but does not unwind DNA or RNA. The R393A protein also binds DNA weaker both in the presence and absence of a non-hydrolyzable ATP analog, suggesting that this Arg-clamp motif functions to tether the protein to the nucleic acid strand on which it is translocating (Lam et al., 2003a).

The second motif characteristic of only helicases from strains of HCV and related viruses forms a loop connecting two β-sheets that extend from domain 2. The β-loop structure is composed of residues Thr430 to Ala452, and a pair of residues, Phe438 and Phe444 and is located in a highly conserved region at the loop's tip. The turn of the loop is composed of NS3 amino acids 438 to 444. At the time, the function of this "Phe-loop" was a curiosity. Kim et al. (1998) had proposed that this loop functions like a DNA binding loop found in ssDNA binding proteins. Alternately, Yao et al. (1997) proposed that Phe438 and Phe444 could pack into a hydrophobic pocket together with Phe531, Phe536, and Trp532, allowing the loop to take on a more structural role. Both Phe438 and Phe444 were altered to Ala to assess these two very different possibilities. Mutagenesis of the Phe's that flank the Phe-loop demonstrates that the loop is not involved in nucleic acid binding. Rather, Phe438 and Phe444 are important both for proper protein folding and for modulating conformational changes leading to the release of DNA upon ATP binding (Lam et al., 2003a).

All helicases crystallized to date contain domains that resemble domains 1 and 2, but none share a domain that resembles domain 3. In some helicases, such as PcrA (Subramanya et al., 1996) and Rep (Korolev et al., 1997), two domains replace domain 3, one which extends from domain 1 (called domain 1B) and one that extends from domain 2 (called domain 2B). In several helicase structures that share domains similar to domains 1 and 2 of HCV helicase, such as the RecQ protein (Bernstein et al., 2003), DnaG (Singleton et al., 2001), and eukaryotic translation initiation factor 4A (Caruthers et al., 2000), domain 3 is missing entirely, suggesting that domain 3 might not be required for HCV helicase movements. This is not the case, however, and although its role in unwinding is only beginning to be understood, domain 3 is clearly essential. Deletion of 97 amino acids from the C-terminus of

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Table 1. HCV helicase mutants.Mutant Motif Phenotype Reference(s)A204V I An, B+, H- Tai et al., 2001K210A I A-, Bn, H- Heilek and Peterson, 1997; Levin and Patel, 1999; Min et

al., 1999; Wardell et al., 1999 K210N I A-, B+, H- Tai et al., 2001K210Q I A-, H- Heilek and Peterson, 1997K210E I A-, Bn, H- Kim et al., 1997b; Chang et al., 2000S231A Ia A+, B+, Hn Lin and Kim, 1999T266A H-, No dimer Khu et al., 2001Y267S H-, No dimer Khu et al., 2001T269A TxGx A-, B-, H- Lin and Kim, 1999D290A II A-, Bn, H- Levin and Patel, 1999; Min et al., 1999; Wardell et al.,

1999D290N II A-, Bn, H- Tai et al., 2001E291A II A-, Bn, H- Wardell et al., 1999; Tai et al., 2001E291Q II A-, Bn, H- Tai et al., 2001C292G II A-, Bn, Hn Kim et al., 1997bC292S II A-, Bn, H- Kim et al., 1997b; Wardell et al., 1999C292A II A-, Bn, H- Tai et al., 2001M288T H-, No dimer Khu et al., 2001H293A II A+, B+, H- Heilek and Peterson, 1997; Kim et al., 1997b; Tai et al.,

2001H293K II A-, B+, H- Tai et al., 2001H293Q II A-, B+, H- Tai et al., 2001T322A III A-, B+, H- Kim et al., 1997b; Tai et al., 2001T324A III A-, Bn, H- Tai et al., 2001H369A IV An, Bn, Hn Frick et al., 2004aH369K IV A+, B+, H- Frick et al., 2004aS370A IV An, Bn, Hn Lin and Kim, 1999Y392A An, B-, H- Paolini et al., 2000R393A Arg-clamp An, B-, H- Lam et al., 2003aT411A V A+, B-, H- Lin and Kim, 1999V432A A-, Bn, H- Paolini et al., 2000; Preugschat et al., 2000; Tai et al.,

2001V432D A-, B-, Hn Kim et al., 2003V432R A+, B+, Hn Kim et al., 2003F438A Phe-loop An, B+, H- Lam et al., 2003aF444A Phe-loop A-, B-, H- Lam et al., 2003aT450I An, B+, H+ Lam et al., 2003bQ460A VI A-, Bn, H- Kwong et al., 2000Q460H VI A-, B+, H- Kim et al., 1997b; Wardell et al., 1999R461A VI A-, Bn, H- Kim et al., 1997b; Kwong et al., 2000R461Q VI A-, B-, H- Tai et al., 2001R462A VI A+, Bn, Hn Kwong et al., 2000

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NS3 results in an inactive helicase (Jin and Peterson, 1995; Kim et al., 1997a). Two key residues in domain 3 are Trp501, which stacks against a nucleic acid base to act like a bookend (Lin and Kim, 1999; Preugschat et al., 2000; Kim et al., 2003), and Glu493, which helps repel nucleic acids from the binding cleft upon ATP binding (Frick et al., 2004a).

MECHANISM OF ACTIONThere is presently no consensus on exactly how the HCV helicase unwinds RNA. Debate about the HCV helicase mechanism continues largely because in some experiments, HCV helicase appears to function as a monomer, but in others it appears to be a dimer or a higher order oligomer. Below, attempts will be made to reconcile this and some other controversies, but first, to understand the intricacies of these molecular models, it will be necessary to review a few fundamental characteristics of all helicases.

Table 1. Continued.R462L VI A-, B-, H- Kim et al., 1997b; Chang et al., 2000G463A VI A-, Bn, Hn Kim et al., 1997bR464A VI A-, Bn, H- Kim et al., 1997b; Min et al., 1999; Chang et al., 2000;

Kwong et al., 2000T465N VI A-, Bn, Hn Kim et al., 1997bG466A VI A-, Bn, H- Kim et al., 1997bR467A VI A-, Bn, H- Kwong et al., 2000 K VI A-, Bn, H- Kim et al., 1997b; Wardell et al., 1999E493K A+, B+, H+ Frick et al., 2004aE493Q A+, B+, H+ Frick et al., 2004aW501A An, B-, H- Lin and Kim, 1999; Paolini et al., 2000; Preugschat et al.,

2000; Tai et al., 2001; Kim et al., 2003W501L An, B-, H- Lin and Kim, 1999W501F An, Bn, Hn Lin and Kim, 1999; Preugschat et al., 2000; Kim et al.,

2003W501E An, B-, H- Kim et al., 2003W501R An, B-, H- Kim et al., 2003An Normal ATPase Hn Normal duplex unwindingA+ Enhanced ATPase H+ Enhanced duplex unwindingA- Poor ATPase H- Poor duplex unwindingBn normal nucleic acid bindingB+ Enhanced nucleic acid bindingB- Poor nucleic acid binding

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BASIC PROPERTIES OF ALL HELICASESAll helicases can be divided into two basic groups. Some form rings that encircle DNA (or RNA) while others, like HCV helicase, do not form rings. Both ring and non-ring helicases, are primarily associated with one strand of a double helix and can be classified based on the polarity of that strand. The protein either shifts from the 3'-end to the 5'-end or from the 5'-end to the 3'-end on the strand to which it is mainly bound. The most common method to diagnose the direction of movement is to determine whether the helicase requires a 5'-ssDNA tail or a 3'-ssDNA tail to initiate unwinding. 5'-3' helicases need a 5'-ssDNA tail, and 3'-5' helicases require a 3'-ssDNA tail. HCV helicase is a 3'-5' helicase (Tai et al., 1996; Morris et al., 2002). As a consequence, if the oligonucleotide bound to HCV helicase in PDB file 1A1V (Fig. 1A) (Kim et al., 1998) represents the strand on which HCV helicase translocates, then the duplex portion of the helix would likely be positioned to the right of the protein in Fig. 1A (see cartoon in Fig. 2). Helicases are thirdly classified based on their evolutionary relationships. Gorbalenya and Koonin have used protein sequence comparisons to classify most helicase families into one of three large superfamilies (Gorbalenya and Koonin, 1993). Non-ring helicases are generally members of helicase superfamily 1 (SF1) or superfamily 2 (SF2), while ring helicases are in superfamily 3 (SF3) or in other families not in the three main superfamilies. HCV helicase is a member of SF2, and like all helicases in SF2, shares conserved motifs I through VI described above.

The ring formed by ring helicases usually is composed of six identical subunits assembled in a head-to-tail manner. The rings surround the strand on which the helicase is translocating and the complementary strand passes outside the ring (Egelman et al., 1995). ATP binds between the subunits, to the head of one subunit and the tail of an adjacent protomer. There are consequently six ATP binding sites per hexameric ring (Singleton et al., 2000). Each subunit of a ring helicase contains a single domain that resembles a domain first seen in the structure of a protein called RecA, which plays a key role in E. coli DNA recombination (Story and Steitz, 1992). In ring helicases, ATP hydrolysis leads to rotation of the RecA-like domains which in turn leads to movements of positively-charged loops that protrude into the center of the ring. The positively charged loops bind DNA (Notarnicola et al., 1995; Washington et al., 1996), and the sequential interaction of the DNA-binding loops with DNA is thought to lead to ring helicase movement (Singleton et al., 2000).

In non-ring helicases, like HCV, there are two RecA-like domains in a single protein subunit, and ATP binds between these subunits. In HCV helicase, domains 1 and 2 fold into similar structures although they share no apparent sequence homology. The core of both domains is composed of a series of beta sheets sandwiched between sets of alpha helices. Both domains 1 and 2 are similar to RecA, form the ATP binding site, and contact DNA. The main structural difference between domains 1

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and 2 is that domain 2 contains two long beta sheets that project towards domain 3 (the Phe-loop discussed above), which are not present in domain 1.

MECHANISM OF ATP HYDROLYSISAlthough the position of ATP bound to HCV helicase has not yet been visualized, the mechanism of its hydrolysis most likely resembles that seen in other helicases. The approximate configuration of ATP in the binding site can be seen by comparing a HCV helicase structure with one of a similar helicase that has been crystallized in the presence of a non-hydrolyzable ATP analog. Fig. 3A shows the results of a structural alignment of HCV helicase (PDB file 1A1V) with the SF2 helicase RecQ bound to ATPγS (PDB file 1OYY) (Bernstein et al., 2003). Shown only are the ATPγS (from 1OYY), the HCV helicase, and its bound oligonucleotide (both from 1A1V). Residues that likely play key roles in ATP hydrolysis are highlighted as sticks.

The configuration of residues at the ATP-binding site depicted in Fig. 3A is reminiscent of that seen in all other helicases that have been studied bound to NTPs (Sawaya et al., 1999; Soultanas et al., 1999; Velankar et al., 1999; Singleton et al., 2000; Bernstein et al., 2003; Gai et al., 2004; James et al., 2004). ATP and a required metal ion cofactor (depicted as Mg2+ in Fig. 3A) normally bind to a helicase in the cleft that separates two adjacent RecA-like domains. The most critical residues for ATP binding arise from the Walker A and B motifs (Walker et al., 1982). The Walker A motif of HCV helicase forms a phosphate binding loop (P-loop) with the conserved Lys210 likely contacting the γ phosphate of ATP. The Walker B motif contains acidic residues that coordinate the positively charged divalent metal cation, which in turn contacts the phosphates of ATP. In the alignment in Fig. 3A, Asp290 seems to be ideally suited to coordinate the catalytic metal. In or near the Walker B motif of helicases and related proteins, there is normally a residue which acts as a catalytic base by accepting a proton from the water molecule that attacks the γ phosphate of ATP. Normally, the catalytic base in this class of enzymes is a glutamate (Goetzinger and Rao, 2003; Orelle et al., 2003), and Glu291 seems to be properly positioned to perform this function.

A more detailed analysis of HCV structures suggests that the roles of particular residues might be somewhat more complicated than assumed above. For example, to function as a catalytic base, the pKa of Glu291 would need to be much higher than that of a typical Glu in a protein. However, electrostatic analysis of all HCV helicase structures reveals that neither Glu291, nor any nearby Glu, has an abnormally high pKa. In contrast, Asp290 has a pKa as high as 10 in some structures and as low as 3 in others. Interestingly, in structures in the open conformation (such as 8OHM), the pKa of Asp290 is low, and in the closed conformation (ex. 1A1V), the pKa of Asp290 is higher than 7, suggesting that Asp290 picks up a proton (like a catalytic

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base) when the protein changes from the open to the closed conformation. Thus, Asp290 could serve as a catalytic base instead of, or in addition to, coordinating the magnesium-ATP complex (Frick et al., 2004a).

Once the water molecule is activated, it acts as a nucleophile, most likely attacking the terminal phosphate of ATP. The pentavalent transition state, where the γ phosphate is bound to 5 oxygen atoms, then breaks down into ADP and inorganic phosphate. In similar ATP hydrolyzing enzymes, the transition state is stabilized by one or more positively charged residues, normally arginines, that function in concert with the lysine of the Walker A motif. In many enzymes, including helicases, these "arginine fingers" are frequently part of adjacent protein subunits. For example, in

Fig. 3. Key residues in HCV helicase. (A) HCV helicase residues likely involved in modulating ATP binding and hydrolysis. The approximate position of ATP bound to HCV helicase is revealed by a structural alignment of HCV helicase (PDB file 1A1V) (Kim et al., 1998) with the SF2 helicase RecQ bound to ATPγS (PDB file 1OYY) (Bernstein et al., 2003) (B) Key residues contacting the oligonucleotide bound to HCV helicase in PDB file 1A1V (Kim et al., 1998).

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small GTPases like Ras, the Arg-finger that activates GTP hydrolysis is part of the GTPase-Activating Protein (GAP) (Ahmadian et al., 1997). In F1ATPase, an Arg-finger on the alpha subunit stabilizes the transition state (Nadanaciva et al., 1999). Recently, it was shown that in ring helicases, the Arg-finger and P-loop are part of different polypeptide chains in the hexamer (Crampton et al., 2004), demonstrating why ring helicases need to oligomerize to cleave ATP. In HCV helicase, several arginines are present in domain 2, and they line the ATP binding cleft. These residues are part of conserved motif IV and include Arg461, Arg462, Arg464, and Arg467. In the model shown in Fig. 3, Arg467 and Arg464 are nearest the phosphates of ATP. Either could rotate even closer to ATP if the cleft between domains 1 and 2 closes. Of the two, only Arg467 is shown because it is of the most interest. Arg467 is methylated by cellular protein arginine-methyltransferase I (Rho et al., 2001). Although it is still unclear how methylation influences helicase activity, such a modification should eliminate all activity if Arg467 acts as an Arg-finger. Site-directed mutagenesis supports this contention. When Arg467 is changed to Lys (Kim et al., 1997b; Wardell et al., 1999) or Ala (Kwong et al., 2000), the proteins do not unwind RNA, and ATPase activity is decreased over 10-fold. An R464A mutant has a similar effect (Kim et al., 1997b; Min et al., 1999; Kwong et al., 2000).

Many of the other conserved residues help to properly position the above groups by forming networks of hydrogen bonds, ionic bonds, and hydrophobic interactions. In addition, some conserved residues help coordinate the rotation of domain 2. Two such amino acids are noted on Fig. 3A. His293 in motif II (domain 1) and Gln460 in motif VI (domain 2) are near each other in many structures and could interact. Kim et al. called these residues "gatekeepers" and propose that they might provide a switch modulating the opening and closure of the cleft between domains 1 and 2 upon ATP binding (Kim et al., 1998). Mutation of either residue has a profound and interesting effect on ATPase. Mutation of Gln460 abolishes detectable ATPase, but an H293A mutation results in a protein with a significantly higher level of ATPase in the absence of RNA, and the protein still unwinds RNA. In the presence of RNA, the H293A mutant hydrolyzes ATP slower than wildtype, to such an extent that RNA appears to inhibit ATP hydrolysis (Kim et al., 1997b). These data further support the idea that rotation of domain 2 is related to ATP hydrolysis, and that domain closure leads to a completion of the active site and hydrolysis of ATP. The question of how ATP hydrolysis is translated into helicase movement on nucleic acid still remains unanswered, however. The first two models that were applied to HCV helicase to explain its movements suggest that either the protein operates as a monomer like an inchworm (Kim et al., 1998) or as a dimer, which rolls along nucleic acid (Cho et al., 1998).

THE INCHWORM AND ROLLING MODELSIf ATP binding and hydrolysis leads to movement of domain 2 relative to domain 1, then this conformational change would in turn impact interactions between residues

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in these two domains with RNA. A close-up of the DNA binding site of structure 1A1V is shown in Fig. 3B, with key amino acids highlighted. Unlike SF1 helicases, which have many interactions with nucleic acid bases (Velankar et al., 1999), most of the contacts occur with protein side chains and the sugar-phosphate backbone of DNA (Kim et al., 1998). One key hydrogen bond is donated from domain 1 residue Thr269, which is part of the conserved TxGx motif, and an analogous interaction arises from motif V residue Thr411 in domain 2. Mutagenesis of either residue affects both RNA binding affinity and unwinding rates (Lin and Kim, 1999). Also noted on Fig. 3B are residues Thr450, Arg393, Trp501, and Glu493, whose roles were alluded to above.

Unlike ring helicases that need to oligomerize to cleave ATP because the Arg-finger is located on the opposite side of a protein monomer relative to the Walker A motif, all the residues necessary for ATP hydrolysis are present in a single polypeptide chain in HCV helicase. Monomeric models for HCV helicase action state that upon rotation, ATP binding leads to a closure of the cleft between domains 1 and 2 by a rotation of domain 2 relative to the rest of the protein, a movement first observed in the structures of Yao et al. (1997). Such conformational changes conceivably could cause the protein to act like an inchworm to move along RNA. Most monomeric models are variations on the "ratcheting inchworm" model first proposed for HCV helicase by Kim et al. (1998). Based on the observation that the oligonucleotide appears to be locked into the binding cleft because a residue in domain 3, Trp501, is stacked against the 3'-terminal base, Kim et al. proposed that ATP binding, and the subsequent closure of the cleft between domains 1 and 2, will lead to a ratcheting of Trp501 past 1 or 2 nucleotides. Consequently, the protein would move towards the 5'-end of the bound nucleic acid. After ATP is hydrolyzed and Trp501 is again locked into place acting as a bookend, the cleft opens and RNA slides through the other side of the protein. Kim et al. proposed that the residue that acts as a 5'-bookend, analogous to the 3'-bookend Trp501, might be Val432 in domain 2 (Kim et al., 1998).

The dimeric models for HCV helicase action are essentially variations on Wong and Lohman's rolling dimer hypothesis that was used to explain the actions of a dimer formed by the E. coli Rep helicase upon DNA binding (Wong and Lohman, 1992). In the rolling dimer, each subunit alternates between a form that prefers to bind ssDNA and a form that preferentially binds a double helix. Switching between the states is modulated by ATP binding and hydrolysis. In theory, both forms are bound to a DNA fork, with one subunit bound to the ssDNA tail, and the other bound to the duplex region. When the trailing subunit changes conformation so that it prefers to bind duplex DNA, it will roll toward the double helix causing the subunit bound to the duplex to wrench one strand away from its complement so that it can then bind the resulting ssDNA (for review see Lohman and Bjornson,

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1996). A modified rolling model was applied to the HCV helicase by Cho et al. (1998), who observed that two HCV helicase monomers could pack in the manner shown in Fig. 1F. Cho et al. called their model a "descending molecular see-saw" and proposed that RNA could thread through a long cleft formed between domains 1 and 2 of adjacent subunits (Cho et al., 1998). However, the later structure by Kim et al. (1998) showing DNA bound in another cleft (Fig. 1A), coupled with the fact that ATP likely binds in the cleft between domains 1 and 2 (Fig. 3), makes such an orientation seem unlikely.

EVIDENCE FOR A FUNCTIONAL MONOMER (THE INCHWORM MODEL)Ever since the HCV helicase portion of NS3 was first purified, it was apparent that it behaved as a monomer and did not need to oligomerize to cleave ATP. Initial studies found HCV helicase to act as a monomer in solution based on gel filtration (Preugschat et al., 1996) and analytical ultracentrifugation (Porter et al., 1998). As discussed above, the monomeric enzyme has all the residues necessary to catalyze ATP hydrolysis, and as a result, no decrease in turnover number (kcat) is observed when HCV helicase is diluted (Levin and Patel, 2002). In contrast, diluting a ring helicase leads to a loss of the ability to hydrolyze ATP at low protein concentrations (Guo et al., 1999). There is also yet no direct structural evidence for dimerization. Although the protein exists as a dimer in the crystallographic asymmetric unit in PDB files 1HEI (Yao et al., 1997) and 1CU1 (Yao et al., 1999), it is a monomer in 1A1V (Kim et al., 1998) and 8OHM (Cho et al., 1998). No evidence has been presented that interfaces seen in PDB files 1CU1 or 1HEI are biologically relevant, and Cho's model (Fig. 1F) is based on crystal packing interactions, not actual observed interfaces (Cho et al., 1998).

Soon after the ratcheting inchworm model was introduced for HCV helicase (Kim et al., 1998), it was tested by several groups using site-directed mutagenesis (Table 1). Most initial interest focused on the residues that act as bookends, or the teeth of the ratchet. Several groups have confirmed the importance of Trp501 in both nucleic acid binding and unwinding (Lin and Kim, 1999; Paolini et al., 2000; Preugschat et al., 2000; Kim et al., 2003). Without a bulky aromatic amino acid at position 501, HCV helicase is unable to unwind RNA (Lin and Kim, 1999; Tai et al., 2001; Kim et al., 2003) but retains some ability to unwind DNA (Kim et al., 2003), albeit more slowly than wild type (Preugschat et al., 2000). The data is less clear regarding the residue that might bookend the 5'-end of the RNA. Kim et al. propose that this residue is Val432 in Domain 2 (Kim et al., 1998), but Paolini et al. have suggested that Tyr392 could play a similar role (Paolini et al., 2000). Some of these reports have suggested that mutation of these residues leads to decreases in helicase activity (Paolini et al., 2000; Preugschat et al., 2000; Tai et al., 2001; Kim et al., 2003).

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More recently, other predictions made by the inchworm model have been tested. One basic prediction is that binding of ssDNA to the cleft separating domain 3 from domains 1 and 2 activates ATP hydrolysis. Supporting this theory, several mutations have been made in the DNA binding cleft that decrease the affinity of the protein for both DNA and RNA and affect rates of ATP hydrolysis (see Table 1) (Kim et al., 1997b; Lin and Kim, 1999; Tai et al., 2001). However, there exists an alternate explanation for such results. The mutant proteins might not fold properly or may be less stable than the wildtype, explaining the decreased binding and unwinding activity. In contrast to these negative results, our lab has recently found that substitution of one of two residues in the DNA binding cleft, His369 or Glu493, enhances binding and lowers the amount of nucleic acid needed to stimulate ATP hydrolysis. For example, an E493K mutant enhances binding to RNA in the presence of ATP by several orders of magnitude. This positive result provides the clearest evidence that DNA/RNA binding to this region activates ATP hydrolysis (Frick et al., 2004a).

Another prediction made by the inchworm model is that the ssDNA bound between domain 3 and domains 1 and 2 is the strand on which the helicase is translocating in a 3' to 5' direction. We have tested this idea by analyzing a helicase in which Arg393 is changed to Ala. Without this Arg-clamp in the DNA binding cleft (Fig. 3B), the protein cannot unwind DNA or displace proteins bound to ssDNA. The R393A protein retains full RNA-stimulated ATPase activity, and still binds ssDNA with the same stoichiometry as wildtype, albeit more weakly. These data provide strong evidence that the protein moves along the strand seen in the crystal structure in a 3' to 5' direction and the duplex region would lie as diagramed in Fig. 2 (Lam et al., 2003a).

The inchworm model lastly predicts that ATP binding should modulate the affinity of the protein for RNA (or ssDNA). Early kinetic studies of nucleic acid stimulation of ATP hydrolysis suggest that, indeed, ATP binding weakens the affinity of the protein for RNA (Preugschat et al., 1996). However, it was difficult to confirm this observation by directly measuring dissociation constants because HCV helicase only weakly interacts with most common non-hydrolyzable ATP analogs. A breakthrough came when Levin et al. found that the presence of BeF3 tightly locks the reaction product, ADP, on the enzyme (Levin et al., 2003). In other systems, BeF3 coordinates like the γ phosphate of ATP, so that ADP(BeF3) is essentially a non-hydrolyzable ATP analog (Xu et al., 1997). Using ADP(BeF3), Levin et al. showed that when ATP binds HCV helicase, affinity for DNA falls by almost two orders of magnitude. More recently, Lam et al. have shown that nucleic acid binding to HCV helicase is pH dependent in the presence of ADP(BeF3) but not in the absence of the analog, demonstrating that a conformational change occurs upon ATP binding (Lam et al., 2004), as is also predicted by the inchworm model.

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EVIDENCE FOR A FUNCTIONAL OLIGOMER (THE ROLLING MODEL)While the evidence that HCV helicase acts as a monomer is convincing, there is also evidence that multiple subunits interact with each other to efficiently unwind RNA. Yeast two-hybrid assays provide the most persuasive evidence that NS3 interacts with itself (Flajolet et al., 2000; Khu et al., 2001). In such experiments, the minimum peptide required for an NS3-NS3 interaction contains only domain 1 residues 162-335. This peptide surrounds conserved motifs I, II, and III (Khu et al., 2001), suggesting domain 1 would interact with domain 1 of another monomer, rather than domain 1 interacting with domain 2 as proposed by Cho et al. (1998) (Fig. 1F). Three residues, which were identified using a reverse two hybrid screen, are critical for dimer formation, Thr266, Tyr267 and Met288 (Khu et al., 2001). Met288 is not conserved and is normally an Ile in all but a few HCV isolates (Fig. 2). Mutations of these residues not only influence dimer formation that can be assayed using gel filtration, but also the ability of the protein to unwind DNA (Khu et al., 2001).

Oligomerization of NS3 seems to be dependent on nucleic acids. Before the yeast two-hybrid data were reported, Levin and Patel demonstrated that DNA aids the ability to chemically crosslink HCV helicase into high molecular weight species (Levin and Patel, 1999). Dimerization of NS3 has also been visualized using analytical gel filtration, but only in the presence of an oligonucleotide (Khu et al., 2001). Nucleic acid binding data can sometimes be fit to models that do not take into account subunit interactions (Porter, 1998b; Porter, 1998a; Porter et al., 1998; Levin and Patel, 2002), but under certain conditions, cooperative models fit the data better (Locatelli et al., 2002; Frick et al., 2004b). Taken together, these data suggest that two or more HCV helicase protomers cooperatively assemble onto ssDNA (or RNA) in a controlled manner.

The latest evidence for oligomerization has emerged from measurements of rates of HCV helicase catalyzed DNA and RNA unwinding. Notably, unwinding rates are not linearly dependent on the amount of protein present in the reaction, but rather, accelerate greatly once a critical protein concentration is reached (Lam et al., 2003a; Frick et al., 2004b). By measuring unwinding under single-turnover conditions, several groups have presented kinetic models explaining this cooperativity (Levin et al., 2004; Serebrov and Pyle, 2004; Tackett et al., 2005). As reviewed elsewhere (Bianco, 2004), these models all take into account the interaction of multiple protomers aligned on a ssDNA (or RNA) strand and attempt to calculate the number of base pairs unwound in a single turnover event (called "step size"). The theory holds that an oligomer would unwind many base pairs (10 or more) in a single event while a monomer would only unwind a few base pairs at a time. Levin et al. (2004) have calculated a step size of 9 base pairs using DNA, and using a long RNA substrate, Serebrov and Pyle have determined that 18 base pairs are unwound by HCV helicase in a single step (Serebrov and Pyle, 2004). These values support a

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rolling dimer model and stand in stark contrast with an older calculation by Porter et al. that only a few base pairs of fluorescently-labeled DNA are unwound by HCV helicase in a single turnover event (Porter et al., 1998).

OTHER MECHANISMS EXPLAINING HELICASE MOVEMENTBecause neither the inchworm nor the rolling model fully explains all elements of helicase action, additional models have been recently proposed. One such model states that HCV helicase acts like a Brownian motor (Astumian, 1997, Levin et al., 2005 #1004). A Brownian motor exploits random movements that constantly occur on the molecular level (Brownian motion) and an asymmetrical path to shift an object in a single direction. As diagrammed in Fig. 4A, collisions that occur between HCV helicase, water, and other small molecules constantly transfer small amounts of momentum to the protein so that it wobbles slightly relative to the RNA to which it is bound. In the absence of ATP, the helicase is constrained in a certain location due to molecular barriers. However, when HCV helicase binds ATP, it releases its grip on RNA by changing conformation so that it is free to move along RNA. Random collisions will then be more likely to transfer enough momentum that the protein clears the barrier constraining it to its original position. The key to the Brownian motor model is the asymmetry of the path on which the motor is traveling. Because the path is asymmetrical in the Brownian motor model, the protein will be more likely to move in one direction than the other, and the net result of many movements will be movement in a single direction. If the path were symmetrical, then the protein would be equally likely to return to the original position as it would be to move to either adjacent position, and the net result would be no movement.

In Fig. 4, this irregularity is depicted as an asymmetrical free energy diagram. If the helicase moves in a 5' direction, then it will likely clear a free energy peak and descend to the base of another valley towards the 5' end of the RNA. On the other hand, if the protein moves toward the 3' end of the RNA by the same amount, it will not clear the peak and will return to the position at which it began the cycle. Thus, even though movements occur randomly in either direction and many molecules will remain in the same location, the net result will be that most molecules will move in the same 3' to 5' direction. This model has been applied to HCV helicase by Levin et al. (2003), who recently observed that HCV helicase has a higher affinity for a partially duplex DNA substrate with 3'-ssDNA tails, than it does for either ssDNA alone, or DNA with a 5'-ssDNA tail. They propose that interaction with the fork of the DNA leads to asymmetry of the free energy diagram (Levin et al., 2005). While movements towards the fork likely play some role in helicase movement, the fact remains that two groups have independently observed translocation of HCV helicase in a 3' to 5' direction in systems containing no duplex portion on the ssDNA substrate (Morris et al., 2002; Lam et al., 2003a). Thus, if the Brownian model holds true for HCV helicase, then all asymmetry in the free energy diagram should be intrinsic to the helicase and the nucleic acid strand on which it is traveling.

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Our lab has proposed another model to explain HCV helicase movement that suggests that HCV helicase utilizes electrostatic forces to move along DNA and RNA (Frick et al., 2004a; Lam et al., 2004). This "propulsion-by-repulsion" model (Fig. 4B) is based on two observations. First, DNA is tightly bound in a pocket

Fig. 4. Two possible mechanisms for HCV helicase translocation on RNA. (A) The Brownian motor model (Levin et al., 2005). In the absence of ATP, HCV helicase is confined in a single location on an asymmetrical path of RNA. When ATP binds, binding releases the protein from RNA, allowing random movement (Brownian motion) to transport the helicase either in a 5' or 3' direction. Because the path is asymmetrical, molecules moving in the 3' direction will return to their original position, whereas molecules moving in the 5' direction will change positions. Net movement will be in a 5' direction. (B) The propulsion-by-repulsion model (Frick et al., 2004a; Lam et al., 2004). ATP binding rotates domain 2 so that a positively charged Arg-clamp (Lam et al., 2003a) moves the RNA so that it clears Trp501, which is holding the RNA in a negatively charged cleft. When ATP is bound, the protein repels RNA past Trp501 so that the protein moves in a 5' direction until ATP is hydrolyzed and the protein returns to its original conformation.

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of the enzyme that is highly negatively charged (see Fig. 1B). Second, release of DNA from the enzyme is pH dependent; the enzyme binds weaker to DNA in the presence of ATP at a higher pH. The first observation hints that there is a potential energy buildup when the protein is locked onto DNA in the absence of ATP. The second observation suggests that ionizable residues come in contact with DNA upon ATP binding. We have shown using mutagenesis that one of these key residues is Glu493 in the ssDNA binding cleft (Frick et al., 2004a). In our model, ATP binding leads to a conformational change such that the nucleic acid bases can clear the Trp501 bookend (Lam et al., 2004). In the absence of ATP, RNA cannot exit the enzyme because it is blocked by Trp501 and clamped in the cleft by the Arg-clamp on domain 2 (Lam et al., 2003a). When ATP binds, domain 2 rotates bringing with it the positively-charged Arg-clamp. The Arg-clamp attracts the negatively-charged phosphodiester backbone so that RNA moves free from the bookend. The negatively-charged RNA is then repelled by the negatively charged binding cleft, so it moves through the protein until ATP is hydrolyzed, and the protein clamps it tightly again. In such a model, the step size of the helicase would depend on the nature of the nucleic acid on which the protein is translocating explaining, in part, why different step sizes have been calculated using different substrates (Porter et al., 1998; Levin et al., 2004; Serebrov and Pyle, 2004).

ROLE OF THE PROTEASE DOMAIN AND NS4AReviewing the HCV helicase literature is perplexing because frequently the data reported in one study differs from that reported in other studies. Rates of ATP hydrolysis, DNA/RNA unwinding, and dissociation constants frequently differ by more than 10-fold. The most likely explanation for such differences (when the same protocol is used) is that most labs utilize different recombinant versions of HCV helicase. Many of these proteins are quite different because they either (1) include different portions of NS3, or (2) have been isolated from different HCV strains. Because full-length NS3 is difficult to express in E. coli, a truncated protein containing only NS3 residues 166-631 is frequently used. However, some studies have used helicase constructs with more or fewer N-terminal NS3 residues. Furthermore, some studies use a helicase lacking fusion proteins, whereas other studies utilize a helicase with a N-terminal or C-terminal His-tags, a T7-tag, a GST-tag, or combinations of multiple tags. Frequently, the tags are not removed before analysis.

We have compared numerous NS3 constructs in our laboratory to try to understand why different studies have reported such different results. Initially, we thought that such variation might be due to intrinsic differences between the HCV genotypes. Our comparison of three helicases isolated using the same procedure from three different genotypes noted some differences, but these tended to be small (less than 2-fold) and did not explain the widely divergent data in the literature (Lam et al., 2003b). We then set out to compare the effect of fusion proteins, which are attached

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to the helicase to aid expression and purification, and found that these modifications led to major changes in activity (Frick et al., 2004b). While modifications to the C-terminus did not affect most assays, modification to the N-terminus did, suggesting that the protease domain and the conformation of the region linking it to the helicase could have a major role in aiding the cooperative assembly of the protein on RNA and in unwinding (Frick et al., 2004b).

In our hands (Frick et al., 2004b), full-length NS3 (with NS4A) unwinds RNA better than versions lacking the protease, but hydrolyzes ATP slower, suggesting that it is a more efficient molecular motor. Some of the effects of the protease could be substituted for by GST or His-tag fusion proteins, but several could not, suggesting that RNA makes specific contacts with the protease region. As discussed above, it is not clear where the complementary strand or the duplex region of RNA interacts with HCV helicase. Nevertheless, an electrostatic analysis of the full-length protein (Fig. 1D) reveals that a positively-charged cleft is formed between the protease and domain 2 of the helicase. Residues in this cleft could tether the protein to the negatively-charged phosphate backbone of RNA. It is possible that a similar cleft could be formed when the protease is replaced with a fusion protein, explaining why such proteins have a higher apparent processivity than the helicase domain alone (Frick et al., 2004b).

Not all other studies have noted as clear differences when the protease domain is removed from NS3. For example, even though Kuang et al. found that a NS3-NS4A complex unwinds RNA better than an isolated helicase domain, they also noted NS3 lacking NS4A is a poor helicase relative to an isolated helicase domain, suggesting that the protease without its NS4A cofactor might actually inhibit helicase movements (Kuang et al., 2004). Similarly perplexing data showing relatively poor helicase activity for full-length NS3 have been reported by others (Heilek and Peterson, 1997; Gallinari et al., 1998). The poor helicase activity of some full-length NS3 constructs could be explained by the conformational flexibility of the protein. In order to cleave the rest of the polyprotein, Yao et al. proposed that the protease domain swings away from the helicase via the flexible linker that connects the two regions (Yao et al., 1999). If this occurs, then the putative RNA binding cleft proposed above would be disrupted and the helicase would more rapidly dissociate from RNA substrates.

A model in which RNA binds a cleft between domain 2 of the helicase and the protease would also provide a plausible role for the NS4A peptide in facilitating helicase action. NS4A could hold the protein in a conformation so that the RNA binding cleft between the protease and helicase remains intact, explaining why some investigators find that a NS3-NS4A complex unwinds RNA better than NS3 alone. For example, Pang et al. (2002) compared the activities of a NS3-NS4A

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complex expressed in insect cells (Sali et al., 1998) with a His-tagged, full-length NS3 protein expressed and purified from E. coli, and found that the NS3-NS4A complex requires less time to form a functional complex on RNA. Based on the structure of the NS3-NS4A complex (Fig. 1C), it is difficult to envision a direct interaction between NS4A and RNA, as has been proposed by others (Silverman et al., 2003). Thus, we prefer a model where NS4A stabilizes the formation of an RNA binding cleft on NS3 (Frick et al., 2004b).

In addition to being a better RNA helicase, we also find that the full-length protein oligomerizes more readily than the truncated protein lacking a protease domain, indicating that the protease domain properly configures the protein for oligomerization. As evidence, we find twice as many protomers of full-length NS3 bound to a single oligonucleotide as recombinant proteins containing the helicase domain only (Frick et al., 2004b). This key observation explains why early studies using isolated helicase domains lacking the protease failed to detect cooperative assembly (Preugschat et al., 1996; Levin and Patel, 2002; Lam et al., 2003a), while later studies using the full-length NS3 often detect oligomers (Khu et al., 2001; Locatelli et al., 2002; Frick et al., 2004b).

HCV HELICASE INHIBITORSBecause compounds that inhibit a helicase encoded by herpes simplex virus (HSV) have been recently shown to moderate disease symptoms (Crute et al., 2002; Kleymann et al., 2002), there has been great interest in finding inhibitors of HCV helicase. Many compounds that inhibit HCV helicase have been reported, and they can be broadly classified as small molecules, nucleic acids, or antibodies. There are, however, many obstacles that must be overcome before developing helicase inhibitors into viable antiviral agents. The main problem will likely be toxicity because the motor domains of HCV helicase are conserved in a vast array of cellular proteins. Consequently, there is more focus on finding inhibitors that bind sites that are not conserved with cellular enzymes, such as the RNA binding site(s) described above and possible allosteric regulatory sites. Even if these inhibitors are never developed into drugs, they should still be useful for elucidating the role of HCV helicase in the viral lifecycle.

SMALL MOLECULESMany of the small molecules that were initially examined as HCV helicase inhibitors were nucleoside analogs. Although nucleoside analogs might also inhibit cellular proteins by interacting with conserved Walker sequences, there is some potential for these compounds because there is a possibility that nucleotides could bind to a second site on the helicase in addition to the conserved Walker site. Such a site could be formed, for example, if the protein oligomerizes and ATP binds to an interface between the RecA-like domains of adjacent subunits. Porter et al. first detected a

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possible second nucleotide binding site when they studied product inhibition in the presence of NaF. In their studies, about two moles of ADP bound per protein monomer (Porter, 1998a). In contrast, as discussed above, when beryllium fluoride is added to the reaction, only one mole of ADP is bound per protomer (Lam et al., 2003a; Levin et al., 2003). One model explaining these data assumes that the helicase functions as a dimer with ATP bound tightly to the interface between domains 1 and 2 and ADP bound more weakly to a second interface. ADP fluoride complexes likely do not resemble the substrate, ATP, as closely as ADP(BeF3), so they might bind both active and allosteric sites. Also in support of a second NTP-binding site, Locatelli et al. have demonstrated that nucleotides bind HCV helicase cooperatively (Locatelli et al., 2002).

There is also some evidence that the second potential nucleotide binding site on HCV helicase is more specific than the nucleotide binding site between domains 1 and 2. The alignment in Fig. 3 reveals few contacts are made between HCV helicase and the sugar or base of an NTP, explaining the observed non-specificity of this site. HCV helicase hydrolyzes all eight canonical nucleoside triphosphates (Preugschat et al., 1996; Wardell et al., 1999; Lam et al., 2003b). The seven other (d)NTPs are competitive inhibitors of ATP hydrolysis (Lam et al., 2003b) and most studies find that they all support unwinding. However, one study found that only some NTPs fuel unwinding with efficiency comparable to that seen with ATP (Locatelli et al., 2001). Other (d)NTPs, particularly dATP, were found to be poor substrates and potent inhibitors of unwinding (Locatelli et al., 2001). Such results can be explained if NTP binding to a regulatory site is more specific than NTP binding to the catalytic site. For example, the regulatory site might only bind dATP but not the NTPs that fail to inhibit unwinding.

Regardless of whether nucleoside analogs will ever be developed into anti-HCV therapeutics, the effects of such compounds on HCV helicase have been extensively studied. Examples include ribavirin triphosphate (Borowski et al., 2001), 5'-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine (FSBI) (Bretner et al., 2004), and ring-expanded ("fat") nucleosides and nucleotides (Zhang et al., 2003). Much of the data has been previously reviewed (Borowski et al., 2002a; Borowski et al., 2002b). Generally, such compounds inhibit only at very low ATP concentrations, and are competitive with ATP, so that under physiological conditions little or no inhibition is observed.

Compounds resembling nucleoside bases have also been reported to be HCV helicase inhibitors. For example, tetrachlorobenzotriazole (TCBT) and tetrabromobenzotriazole (TBBT) were recently analyzed as helicase inhibitors. Both compounds inhibit unwinding catalyzed by helicases from related viruses (such as

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West Nile virus) with IC50's in the low micromolar range, but only TBBT inhibits RNA unwinding by HCV helicase (IC50 ~60 μM). Neither compound inhibits helicase-catalyzed ATP hydrolysis (Borowski et al., 2003), but it is still not clear whether these compounds bind a true allosteric site or if they inhibit unwinding by non-specific interactions with the nucleic acid substrate.

Many groups have reported non-nucleoside based inhibitors of HCV helicase, primarily in the patent literature. These compounds include a piperidine derivative, heterocyclic carboxamide, antracycline antibiotics (Borowski et al., 2002b), paclitaxel, trifluoperazine (Borowski et al., 2002a), and aminophenylbenzimidazole derivatives (Phoon et al., 2001). Many of these compounds intercalate in nucleic acids and likely act via that non-specific mechanism. Whether or not any of these compounds or other small molecules decrease HCV replication measured using replicons or animal models is still yet to be reported.

NUCLEIC ACID BASED INHIBITORSOne of the unique properties of HCV helicase is that, unlike other helicases, the protein binds RNA and DNA in a sequence specific manner. Even the first studies of the protein noted that HCV helicase has a distinctive nucleic acid stimulation profile (Suzich et al., 1993). This means that ATP hydrolysis is stimulated by some nucleic acid polymers much better than it is stimulated by others. The range is quite dramatic. Poly(G) RNA does not stimulate at any measurable level, and poly(U) RNA (or DNA) stimulates best (up to 50-fold). Interestingly, differential stimulation is not entirely due to differences in binding affinity. Direct binding assays confirm that poly(U) binds HCV helicase tighter than polymers composed of the other bases (Gwack et al., 1996), but at saturating nucleic acid concentrations, not all sequences support the same maximum rate of ATP hydrolysis, suggesting that the protein assumes different conformations when bound to different sequences (Lam et al., 2003b).

Previously, I have proposed that HCV helicase nucleic acid specificity stems from interactions between conserved amino acids in the RNA binding cleft, particularly Trp501, Glu493, and Asn556, with nucleic acid bases, either directly or through water molecules (Frick, 2004). As evidence supporting this hypothesis, mutation of Trp501 has been reported to result in a protein that is more efficiently stimulated by poly(C) than poly(U) RNA (Lin and Kim, 1999), and there is a change in the nucleic acid stimulation profile when Glu493 is substituted by another amino acid (D. N. Frick and R. S. Rypma, unpublished results). RNA specificity has also been proposed to play a role in directing the helicase, and possibly the entire HCV replication complex, to certain regions of the viral genome. For example, HCV helicase specifically binds both to the 3'-UTR and the 3'-end of the negative strand viral transcript (the complement of the 5'-UTR). This might be necessary during the viral lifecycle to allow the NS5B polymerase to synthesize RNA in these regions that contain stable secondary structures (Banerjee and Dasgupta, 2001).

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Nucleic acid interactions with HCV helicase depend not only on the base composition but also on the composition of the nucleic acid backbone. It is widely recognized that HCV helicase unwinds a DNA duplex more efficiently than an RNA duplex. The biological reason for this, if there is one, is still a mystery because there is no DNA stage in the viral lifecycle, and replication likely occurs on the endoplasmic reticulum (Wolk et al., 2000). However, some reports have detected NS3 in the nucleus (Muramatsu et al., 1997; Errington et al., 1999), where the helicase could modify host gene expression (Sakamuro et al., 1995). Whereas loss of the 2'-OH group from RNA permits the helicase to unwind substrates faster (DNA is unwound faster than RNA), adding a methyl group to this position (2'-O-methyl RNA) weakens helicase interaction with RNA and prevents unwinding (Hesson et al., 2000). The effects are strand specific in that the helicase only appears to sense the chemical composition of the strand with the 3'-overhang (the longer strand in the helicase substrate). Composition of the shorter strand does not affect unwinding rates as drastically, suggesting that interactions are made primarily with the nucleic acid sugars of only one strand of the helicase substrate. When the longer strand (with the 3' overhang) is DNA, the shorter strand can be composed of RNA, 2'-O-methyl RNA, morpholino-DNA, or phosphorothioate-DNA without affecting unwinding (Hesson et al., 2000; Tackett et al., 2001; Pang et al., 2002). However, if the shorter strand is composed of peptide nucleic acid (where a N-(2-aminoethyl)glycine backbone replaces the deoxyribose phosphates), then unwinding is slower than with natural substrates (Tackett et al., 2001). Whether peptide nucleic acids are poor substrates because of the lack of specific interactions with HCV helicase, or simply because they form more stable duplexes, is still unclear (Tackett et al., 2001).

Two groups have tried to exploit the nucleic acid specificity of HCV helicase with a goal of developing RNA-based inhibitors. Both groups have used SELEX (systematic evolution of ligands by exponential amplification) to find RNA aptamers that tightly bind HCV helicase. In the SELEX procedure, an RNA library is screened for sequences that bind a macromolecule. Only those sequences that bind tightly are amplified to create a new library, and the selection process is repeated with the new library. Although directly using RNA as an antiviral drug will be challenging because of its cellular instability, the information derived from aptamer studies could be used to make more stable derivatives or by delivering RNA directly to infected cells using gene therapy (for more on anti-HCV nucleic acids see Chapter 18).

One set of aptamers specific to HCV helicase was generated by modifying aptamers that bind tightly and inhibit the NS3-NS4A serine protease. Such aptamers were found to bind truncated NS3 lacking the helicase domains using SELEX. They all share the conserved sequence GA(A/U)UGGGAC (Fukuda et al., 2000), bind NS3 protease over one thousand times tighter than random RNA sequences and

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are effective at inhibiting the HCV NS3 protease (Fukuda et al., 2000; Nishikawa et al., 2003). When positions Arg130, Arg161, and Lys165 are substituted with Ala, the aptamers no longer bind NS3, suggesting that they interact near these NS3 residues, which are located in the region that links the protease to the helicase (Hwang et al., 2000). To create an aptamer that inhibits protease and helicase activity of NS3, a 14-mer uridine tail was added to one of the most effective HCV protease-binding RNA aptamers. The new, longer aptamer interacts with both the protease and helicase domains of the full-length NS3 protein (Fukuda et al., 2004), binds to the helicase portion of NS3 with high affinity (Kd ~4 nM) (Fukuda et al., 2004), and inhibits the NS3 helicase activity with an EC50 of ~500 nM. The same group has recently reported a new "advanced dual functional" aptamer in which another aptamer, selected for helicase binding (Nishikawa et al., 2004), is tethered to a protease-binding aptamer using a poly(U) linker. This new aptamer is about five times more effective than either aptamer when they are not covalently linked (Umehara et al., 2005).

A second group has also selected for aptamers using the helicase portion of NS3 as the bait in the SELEX procedure (Hwang et al., 2004). This aptamer (called SE RNA) folds to form four stem loops with GC pairs that are similar to the stem loop located at the 3'-terminal of the negative strand HCV RNA. This observation suggests that the SE aptamer might bind the helicase in a similar manner as the stem loop located at the 3'-terminal of the negative strand HCV RNA (Banerjee and Dasgupta, 2001). SE RNA binds the HCV helicase tightly (Kd ~990 pM), efficiently competes with poly(U), stimulates ATP hydrolysis, and potently inhibits RNA unwinding (IC50 ~12.5 nM). When delivered to human liver cells (Huh 7) infected with HCV replicons, the SE aptamer slows HCV RNA synthesis, and interestingly, labeled SE aptamers can also be used as a diagnostic tool to detect the NS3 protein in cells from HCV patients (Nishikawa et al., 2004).

ANTIBODIESThe third, and possibly most ambitious, method that is currently being explored to inhibit HCV helicase is to generate antibody-like molecules that, when expressed intracellularly, will bind and inhibit HCV helicase activities. Almost all HCV patients produce antibodies directed against the NS3 protein, and the vast majority of these bind to the helicase portion of the protein (Chen et al., 1998). Several groups are working toward the goal of introducing recombinant antibodies into cells for "cellular immunization," a procedure which has been used experimentally with HIV (Goncalves et al., 2002). In this approach, HCV infected cells are transfected with a gene expressing a portion of an antibody selected for reactivity with NS3. One method is to use single chain fragment (ScFv) antibodies. A ScFv is composed of the immunoglobulin heavy chain variable domain connected to the variable region of the light chain by a polypeptide linker. Such a molecule can be constructed using

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PCR. The other principal method uses an antibody fragment (Fab), which contains the complete light chain and the variable and first constant domains of the heavy chain. A Fab is larger and usually more stable than a ScFv.

To construct a ScFv, immunoglobulin specific PCR is first used to construct a library of human antibody fragments using plasma cells from HCV patients as the PCR template. To identify which antibodies react with HCV helicase, the fragments are fused to a bacteriophage coat protein for phage display, and phages with a high affinity for HCV helicase are purified. Tessman et al. have used this technique to isolate a series of high affinity ScFv's that specifically interact with HCV helicase (Tessmann et al., 2002). ScFv's that bind HCV helicase have also been constructed by splicing together the variable domains of monoclonal antibodies (Zhang et al., 2000; Sullivan et al., 2002), and after expression and purification, several of these recombinant proteins inhibit HCV helicase-catalyzed DNA unwinding (Sullivan et al., 2002; Artsaenko et al., 2003). One particular ScFv consists of the variable regions of the human monoclonal antibody CM3.B6, which recognizes an epitope that spans conserved SF2 helicase motifs IV and V (Mondelli et al., 1994). The CM3.B6 ScFv has been expressed in HCV infected hepatocytes (HepG2 cells), immunoblots of which reveal an intra-cellular interaction between the antibody and NS3. HCV RNA synthesis within primary hepatocytes infected with HCV is also reduced by 10-fold when the cells contain a vector carrying the CM3.B6 ScFv gene (Sullivan et al., 2002).

Phage display has also been used to isolate an anti-HCV helicase Fab from a patient infected with HCV genotype 1b. Prabhu et al. have isolated this human Fab, called HFab-aNS3, and demonstrated that it has HCV antiviral activity (Prabhu et al., 2004). HFab-aNS3 recognizes an epitope that spans motifs I to V of the protein, and when purified and pre-incubated with HCV helicase, HFab-aNS3 abolishes detectable DNA unwinding. Intracellular expression of HFab-aNS3 within replicon-transfected Huh 7 cells suppresses NS3 protein expression and significantly inhibits viral RNA synthesis of both subgenomic and full-length HCV replicons (Prabhu et al., 2004).

CONCLUSIONS AND FUTURE DIRECTIONSHCV helicase has attracted the attention not only of researchers interested in developing novel antiviral drugs, but also those studying how proteins interact with nucleic acids. As one of only three helicases that have been crystallized bound to an oligonucleotide, HCV helicase has become one of the best model proteins to study how helicases unwind duplexes and move on DNA and RNA. Different theories on how chemical energy stored in ATP is transformed into the mechanical force necessary to move a protein from one position to another are currently hotly debated, and many have been tested using the HCV NS3 protein. Obviously, work will continue until HCV helicase's precise mechanism of action is defined.

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While it is still uncertain if HCV helicase functions using an inchworm, rolling, Brownian, or electrostatic mechanism, extensive work has uncovered its basic properties and the roles of several key residues. ATP binds HCV helicase between two RecA-like domains, causing a conformational change that leads to a decrease in the affinity of the protein for nucleic acids. Key residues contacting ATP include Lys210, which likely coordinates the phosphates, Asp290, which could coordinate a divalent metal ion, Glu291, which might act as a catalytic base, and one or more arginines on the adjacent domain. One strand of RNA binds in a second cleft formed perpendicular to the ATP-binding cleft and its binding leads to stimulation of ATP hydrolysis. RNA and/or ATP binding likely causes rotation of domain 2 of the enzyme relative to domains 1 and 3, and somehow this conformational change allows the protein to move like a motor. Key residues involved in RNA binding include Trp501, which locks the protein in position in the absence of ATP, and Glu493, which repels RNA when ATP binds.

Additional structural and mechanistic work may or may not be necessary in order to develop helicase inhibitors into antiviral drugs. For example, much less is known about the structure and mechanism of action of the herpes simplex virus helicase that is the target of newly developed antiviral drugs (Kleymann, 2004). In contrast, the biological role of the HSV enzyme in viral replication is much more clearly defined than that of the HCV helicase. The HSV helicase targeted by antiviral drugs is needed to coordinate RNA primer synthesis on the lagging strand while the double helix is unwound (Boehmer and Lehman, 1997). All that is firmly established about the biological function of HCV helicase is that if its ability to hydrolyze ATP is abolished, the virus will no longer infect chimpanzees (Kolykhalov et al., 2000).

Clearly, the biological role of HCV helicase needs to be investigated in more detail. Perhaps the many inhibitors or site-directed mutants of HCV helicase could be used to design experiments elucidating its role using the replicon system. If the helicase is only needed to unwind duplex RNA formed after RNA synthesis, then replicons with a defective helicase should still synthesize small amounts of both the polyprotein and negative sense RNA. However, when an antibody against HCV helicase is co-expressed in cells expressing HCV replicons, there is not only diminished positive strand synthesis but also less synthesis of HCV negative strand RNA and HCV proteins, suggesting that the helicase plays numerous complex and important roles in the viral lifecycle (Prabhu et al., 2004). Given the propensity for HCV helicase to unwind DNA, issues regarding possible roles of the NS3 protein in manipulating cellular DNA should also be more thoroughly examined.

While presently it appears that HCV protease and HCV polymerase inhibitors will be developed as the next generation of anti-HCV drugs, compounds inhibiting

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HCV helicase might also someday prove therapeutically useful. Both NS5B and NS3 protease have clearer roles in HCV replication, and unlike helicases, the mechanisms of serine proteases and RNA polymerases have been understood for decades. Consequently, it is not surprising that HCV helicase inhibitor development lags behind that for the other HCV enzymes. As long as its mechanism and role in replication are not clearly understood, development of antiviral drugs targeting HCV helicase will remain difficult. Nevertheless, rapid progress is being made in the helicase field, and it will not be surprising if HCV helicase inhibitors someday enter clinical trials.

ACKNOWLEDGEMENTSThis work was supported by National Institutes of Health grant AI052395. I am grateful to Angela M. I. Lam and Ryan S. Rypma for reviewing the manuscript and Christopher M. Frenz for help preparing the figures.

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The HCV Non-structural Protein 4B

Chapter 8

HCV NS4B: From Obscurity to Central Stage

Ella H. Sklan and Jeffrey S. Glenn

ABSTRACTThe hepatitis C virus (HCV) non-structural 4B (NS4B) protein is a 27kDa hydrophobic protein which for many years was characterized mainly as a protein of unknown function. Recently, however, information about the protein and its involvement in mediating various viral activities and effects on host cells is beginning to accumulate. NS4B has been implicated in modulation of NS5B's RNA dependent RNA polymerase activity and various host signal transduction pathways, a possible role in HCV carcinogenesis, impairment of ER function, and regulation of both viral and host translation. Perhaps most significant, NS4B has recently been found to be responsible for the formation of a novel intracellular membrane structure, termed the membranous web, which appears to be the platform upon which viral replication occurs. Specific domains within NS4B have been identified which likely underlie the mechanisms employed by NS4B to mediate many of the preceding functions. As such, these domains which include an amphipathic helix and nucleotide-binding motif represent attractive targets for new antiviral strategies.

INTRODUCTIONWith the cloning of HCV (Choo et al., 1990) it was possible to deduce many of the expected protein products encoded in the viral genome. Among these was a 261 aa protein now known as NS4B. Unlike several other predicted protein products of the HCV polyprotein, no obvious functions could be immediately ascribed to NS4B. For years, NS4B essentially remained a membrane-associated protein characterized mainly by a lack of known function. Recently, however, considerable information concerning NS4B as begun to accumulate. These efforts have both led to a realization of NS4B's importance to the viral life cycle and yielded attractive new targets for antiviral drug development. This chapter will attempt to review these developments and speculate on some of the future directions in this increasingly exciting field. After discussing NS4B genesis, localization and topology, NS4B's relationship with a specialized type of membrane will be addressed. A survey of various NS4B properties and functions will follow, including NS4B's role in the induction of a novel type of membrane structure which represents the candidate site for HCV replication. Finally, we will focus on features within NS4B which may underlie the mechanisms employed by NS4B to mediate its associated functions.

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PROTEOLYTIC GENERATION OF NS4BThe HCV genome is translated from a single ~3000 amino acid-long open reading frame into a large polyprotein that is processed both co- and posttranslationally by a combination of host and viral proteases (see Fig 1). Cleavages generating the HCV structural proteins result from the sequential action of the endoplasmic reticulum (ER) resident enzymes signal peptidase (Hijikata et al., 1991) and signal peptide peptidase (Weihofen et al., 2002). Processing of the non-structural protein region is mediated by two virus-encoded proteases: The zinc-stimulated NS2-3 autoprotease cleaves at the NS2/3 junction, and the NS3 serine protease is responsible for liberating the remaining downstream non-structural proteins (Lindenbach and Rice, 2001). Efficient activity of the NS3 protease requires NS4A, a 54 amino acid-long protein which acts as a cofactor for processing at the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites (Bartenschlager et al., 1994; Failla et al., 1994; Tanji et al., 1995). Stable partial cleavage products that include NS4B can also be detected during the cleavage process (see Fig. 1 Bartenschlager et al., 1994). In other positive strand RNA viruses, such partial cleavage products have independent activities, distinct from those associated with the completely processed individual proteins (LaStarza

Fig. 1. Kinetics and possible partial cleavage products of HCV polyprotein processing. The first cleavage event as indicated by the appearance of partial cleavage products is between NS3 and NS4A. NS4A/B cleavage appears to be delayed as shown by the presence of the NS4A/B intermediate and the slow production of NS4B. Two cleavage pathways seem to operate at the NS4B/5A site: A rapid cleavage with production of the NS4A-B intermediate and a slow cleavage as indicated by the presence of the relatively stable NS4A-4B-5A intermediate. Emphasis is placed on the generation of NS4B. Other cleavage products are observed in cis and trans reactions. See Bartenschlager et al., 1994, and Lin et al., 1994, for details.

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et al., 1994; Parsley et al., 1999). As described further below, there is evidence that at least for one partially-processed precursor including NS4B, HCV may also make use of such a strategy which increases the repertoire of functionally distinct protein-encoded activities. Moreover, although many studies of NS4B examine the effects of NS4B alone, NS4B may also function as part of multiprotein complexes with NS4A and NS5A, with or without NS3 and NS5B (Lin et al., 1997; Neddermann et al., 1999) (Fig. 1).

SUBCELLULAR LOCALIZATION AND TOPOLOGYInitial studies of NS4B attempted to determine its subcellular localization as a first step towards the understanding of its function. Indirect immunofluorescence and green fluorescent protein (GFP) fusion experiments determined that NS4B is cytoplasmically-localized in the perinuclear region where it adopts chickenwire-like and speckled patterns typical of a membrane-associated protein (Kim et al., 1999; Selby et al., 1993). Later Hugle et al. (Hugle et al., 2001) combined the use of several methods, including specific antibodies and confocal analysis, to show that NS4B was localized to the ER, where it colocalized with the other HCV nonstructural (NS) proteins. This localization has been observed when the protein was expressed either alone or in the context of HCV's other NS proteins, as well as in cells harboring HCV replicons (El-Hage and Luo, 2003; Mottola et al., 2002). Lundin et al. (Lundin et al., 2003) confirmed these localization results and reported that the speckle or foci-like structures not only contained ER markers, but that they tended to be both larger and more common the longer the cells are allowed to express recombinant NS4B.

A recent study examined the localization of NS4B in live cells using a chimeric NS4B-GFP fusion (Gretton et al., 2005). NS4B appeared to be distributed in a thread-like pattern, consistent with ER localization, and at small foci similar to those described by others (Gosert et al., 2003; Moradpour et al., 2004). The authors termed these foci membrane-associated foci (MAFs). The mobility of NS4B in ER membranes and MAFs was assessed using fluorescence recovery after photobleaching (FRAP) experiments. In these experiments fluorescent molecules in a defined area are irreversibly photobleached by a high-power laser. Subsequent diffusion of non-bleached molecules into the bleached area leads to a recovery of fluorescence. Fluorescence intensity in selected regions in live cells expressing the NS4B-GFP protein was measured before and after photobleaching. A topologically related GFP-tagged DNase X was used as a control. NS4B was determined to have reduced mobility in MAFs compared with the ER membrane suggesting that NS4B is likely to form different interactions on MAFs and the ER.

In vitro transcription–translation experiments performed in the presence of microsomal membranes revealed that targeting to the ER membrane is

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cotranslational. Using classical membrane extraction and proteinase protection assays it was shown that the majority of the protein is cytoplasmically oriented but it was not possible to demonstrate the presence of transmembrane or lumenal fragments (Hugle et al., 2001). This latter finding seemed somewhat puzzling in light of other viral NS4B proteins having at least one transmembrane domain (TMD) and that various computer predictions have estimated that HCV NS4B has several TMDs. The inability to experimentally detect such TMDs could have been for a variety of technical reasons and the lack of available antibodies to facilitate the detection of such fragments.

Another approach to probing NS4B topology involved introducing canonical glycosylation sites at various positions within NS4B. Here the rationale was that the host cell enzymes responsible for glycosylation at such sites are exclusively located within the ER lumen. Thus, those sites contained within NS4B segments which are truly intralumenal would be expected to undergo glycosylation. Only two of the predicted TMDs connected by a putative ER-lumenal loop could be supported experimentally using this method (Lundin et al., 2003). It is possible, however, that the extra amino acids introduced into NS4B in order to insert the target glycosylation sites may have caused unanticipated deleterious changes to NS4B. Another study found that all predicted TMDs could be deleted without impairing NS4B's ability to associate with membranes (Elazar et al., 2004). Within the remaining segments of NS4B, the authors detected a predicted amphipathic alpha helix domain at the protein's N-terminus which was necessary for conferring membrane association upon the mutant NS4B devoid of all TMDs (see more details below). Nevertheless, the authors still favored a predicted NS4B topology containing TMDs, and suggested that the membrane-associating function of the amphipathic helix was more likely to mediate other membrane-associated functions of NS4B beyond simple anchorage to membranes (Elazar et al., 2004). One such function may be to mediate a topologic change of NS4B proposed to occur based upon an unexpected finding of the above-mentioned glycosylation studies. Indeed, Lundin et al. (Lundin et al., 2003) observed that a glycosylation site introduced into the NS4B N-terminal segment—predicted to be cytoplasmically-oriented—was glycosylated in a manner to suggest that it was translocated into the lumen in a fraction of the NS4B molecules. Such cases of alternate topologies have been described in other viruses such as the hepatitis B virus L envelope protein (Bruss et al., 1994) or the M protein from transmissible gastroenteritis corona virus (Escors et al., 2001). The NS4B proteins of the yellow fever and dengue viruses (Cahour et al., 1992; Lin et al., 1993) also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in HCV NS4B. This potential shared topology might support the idea of a common function for NS4B in Flaviviridae (Lundin et al., 2003).

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Although the above studies clearly show that NS4B behaves as a membrane-associated protein, the precise nature of this association awaits further definition. A variety of topologies with respect to the ER membrane have been proposed, with NS4B predicted to have between 4-6 TMDs. The N and C termini are expected to be (at least initially) located in the cytoplasm since they are generated by the cytoplasmic NS3 protease (Hijikata et al., 1993; Wolk et al., 2000). Part of the uncertainty stems from the fact that the computer algorithms used to predict these topologies are derived from a databank of solved structures which contain inadequate numbers of membrane proteins.

NS4B AND LIPID RAFTSLipid rafts are cholesterol- and sphingolipid-rich microdomains of cellular membranes which are operationally-defined by their resistance to solubilization with certain non-ionic detergents at 4°C (Cohen et al., 2004; Pike, 2004). Classically, these detergent-resistant membrane microdomains and associated proteins "float" to the low density upper fractions when subjected to density gradient ultracentrifugation—hence the designation of rafts. Lipid rafts are known to play important roles in diverse processes such as signal transduction and protein sorting (Simons and Toomre, 2000; Slimane et al., 2003). Rafts are also exploited by an increasingly recognized number of viruses as portals for viral entry or assembly and release (Campbell et al., 2001; Cuadras and Greenberg, 2003; Manes et al., 2003).

The first indication that HCV might also exploit lipid rafts was the demonstration that both viral proteins and RNA could be detected in low density membrane

fractions resistant to solubilization with 1% NP-40 at 4°C (Shi et al., 2003). Although their analysis was limited to NS5A and NS5B, these results suggested that the membranes upon which HCV RNA replication occurs may be lipid rafts recruited from intracellular membranes.

A more detailed analysis revealed that, when expressed together, NS proteins 3 through 5B could all be found in lipid raft fractions (Shi et al., 2003). When expressed individually, however, only NS4B was completely associated with lipid rafts (Gao et al., 2004). Therefore, NS4B, may be the key protein responsible for binding to lipid rafts first and thereby enabling the recruitment or anchoring of other NS proteins in order to form potential replication complexes.

This replication complex-harboring raft may be the same or different from that with which the structural HCV core protein associates (Matto et al., 2004). Moreover, although the raft targeted by NS4B shares biochemical features similar to those of classical plasma membrane rafts, there appears to be some important differences. For one, the steady-state cytoplasmic speckled staining pattern of NS4B is very

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different from the peripheral surface pattern typical of plasma membrane-based rafts (Matto et al., 2004). Second, a significant amount of the raft-resident HCV NS proteins and RNA are protected from digestion with exogenously added protease and nuclease (Aizaki et al., 2004). This protection is lost upon treatment with raft-solubilizing conditions. Taken together, these data suggest NS4B targets the replication complex components to a specialized raft compartment which is not directly contiguous with the host cell cytosol. It is tempting to speculate that this compartment overlaps with the membranous web (to be described later) and that the vesiculation and physical conformation of the latter helps provide some of the observed protection from experimental nucleases. Similar protection may be provided against host intracellular antiviral mechanisms (Barber, 2001).

THE IMMUNOLOGICAL EFFECTS OF NS4BNS4B is recognized as a target by both the humoral and cellular arms of specific immunity. Historically, NS4B sequences were contained in both the seroreactive clone used to originally isolate the first fragment of the HCV genome (Choo et al., 1989) as well as in the recombinant antigen used in the first generation of anti-HCV commercial assays (Conry-Cantilena, 1997). Indeed some of the most diagnostically relevant antigenic epitopes have been found to reside within NS4B (Chang et al., 1999; Masalova et al., 2002; Rodriguez-Lopez et al., 1999) and this helps explain why NS4B has since been successfully used as an antigenic target in various commercial diagnostic tests for the detection of HCV antibodies in the serum of patients with HCV infection. These highly antigenic properties might prove beneficial for therapeutic purposes as well, such as using NS4B-derived peptides to elicit an antiviral cytotoxic T lymphocytes (CTL) response.

To further characterize some of the mechanisms by which HCV induces an inflammatory and immune response, Kato et al. (Kato et al., 2000) used HCV viral protein– expression vectors cotransfected into mammalian cells with reporter vectors having a luciferase gene driven by various cis-enhancer elements from 5 intracellular signaling pathways associated with cell proliferation, differentiation, and apoptosis. Although core had the strongest effects, NS4B also significantly activated the NF-κB–associated signal. Because the NF-κB pathway is known as an inducer of inflammatory and immune responses, it was therefore suggested that HCV core and NS4B proteins might modulate the production of various cytokines and inflammatory responses in HCV-infected liver.

One of these cytokines appears to be interleukin-8 (IL-8). Indeed, serum IL-8 levels have been shown to be elevated in patients infected with HCV (Polyak et al., 2001b) and IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells (Kadoya et al., 2005). Expression of NS4B, (and to a lesser extent NS4A) alone were each found to significantly

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transactivate the IL-8 promoter, resulting in enhanced production of IL-8 protein. The mechanism of IL-8 induction may be via NS4B's effect on NF-κB, as the IL-8 promoter contains a binding site for NF-κB. Because NS5A has also been implicated in the induction of IL-8 (Polyak et al., 2001a), there would appear to be multiple mechanisms whereby such induction can be induced by HCV.

As many cases of HCV are refractory to interferon (IFN) treatment, potential mechanisms underlying HCV resistance to IFN have been the subject of intensive investigation. One approach for studying such HCV resistance to IFN, centered around developing IFN-resistant HCV replicons. For that, cells harboring HCV replicons were subjected to a prolonged low-dose treatment with IFNs. Total RNA derived from these IFN-treated replicon cells was then electroporated into naïve cells and individual cell lines harboring HCV replicons with an IFN-resistant phenotype were isolated. Here too, a possible role of NS4B was found. Indeed, sequencing of the replicons contained in these cell lines revealed that they all shared a single common amino acid substitution in NS4B (Q1737H) which might at least partially explain their IFN-resistant phenotype (Namba et al., 2004). These results should be interpreted with caution since cells cured from the replicon by cyclosporin A, continued to show resistance to IFN suggesting that a host factor(s) rather than replicon RNA(s) could have contributed to the IFN-resistant phenotype. Moreover, direct demonstration that introduction of the Q1737H mutation into a wild type replicon can confer IFN-resistance is still pending.

MODULATION OF NS5BS' RNA-DEPENDENT RNA POLYMERASE ACTIVITYIt was recognized early on that HCV NS5B encodes the virus' RNA-dependent RNA polymerase activity required for HCV replication. Many questions about how this activity is controlled, however, remain unanswered.

To study the potential influence of NS3 and NS4B proteins on the priming activity of NS5B, recombinant proteins were generated and introduced into an assay for NS5B's RNA-dependent-RNA-polymerase (RdRp) activity on a template corresponding to the minus strand 3'-untranslated region ((-)3'-UTR) (Piccininni et al., 2002). Physical interactions between NS3 and NS5B as well as between NS3 and NS4B were demonstrated. Both recombinant NS3 and NS4B proteins were also found to modulate NS5B's RdRp activity, but in distinct ways: NS3, via its helicase function, facilitated NS5B activity, whereas this effect was antagonized by the addition of NS4B. These results provide additional evidence that NS4B can function as part of a multi-protein replication complex. Although this data suggests that NS4B might be a negative regulator of NS5B activity in vitro, this need not be the case in vivo where additional regulatory factors may be operative.

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EFFECTS OF NS4B ON TRANSLATIONSince many viruses are known to interfere with host translational mechanisms, possible inhibitory effects of HCV proteins on cellular protein synthesis were analyzed using a transient expression system. The core protein, NS4A and NS4B, but not NS3, NS5A or NS5B, inhibited the expression of cell cycle regulator protein p21/Waf1/Cip1/Sdi1 (p21/Waf1) (Florese et al., 2002). There were no significant differences in steady-state p21/Waf1 mRNA levels, as demonstrated by RT-PCR and Northern blot analyses, suggesting the possibility of post-transcriptional inhibition. That the inhibitory effect of NS4B may be at the level of translation was suggested by in vitro translation assays which revealed inhibited synthesis of p21/Waf1 protein when co-translated with NS4B RNA. A similar inhibitory effect of NS4B on the expression of RNaseL was detected although the magnitude appeared to be somewhat smaller. It should be noted that a possible contribution of degradation has not been ruled out.

Using a bicistronic reporter plasmid, the effects of HCV proteins on both cap-mediated (host) and internal ribosome entry site (IRES)-mediated (virus) translation were simultaneously monitored (Kato et al., 2002). In this system, the Renilla luciferase is translated in a cap-dependent manner, while the firefly luciferase is translated from the HCV IRES in a cap-independent manner. Both activities were decreased with the expression of NS4A and/or NS4B proteins suggesting that NS4A and NS4B proteins inhibited both cap-dependent translation and cap-independent translation from HCV IRES. It was suggested that the latter might be a viral self-regulation mechanism limiting the amount of viral protein. In contrast, using a similar bicistronic reporter gene construct, IRES-mediated translation was found to be specifically upregulated in HCV replicon cells (He et al., 2003). No such enhancement was observed when the IRES from either poliovirus or EMCV were substituted for the reporter construct's HCV IRES, suggesting specificity for HCV. Transient expression of individual HCV non-structural proteins in combination with the dual-luciferase reporter construct containing the HCV IRES showed that NS5A and to a lesser extent NS4B could stimulate HCV IRES activity, although the effect was less dramatic than in the context of the entire subgenomic replicon. Reduced phosphorylation levels of both eIF2a and eIF4E were observed in the replicon cells. In the absence of further mechanistic details whereby NS4B may mediate its effects on translation, it is difficult to fully reconcile the above-detailed differences observed by different investigators. Perhaps such discrepancies are due to differences in duration and levels of expression of NS4B, or the presence of a critical host cell factor.

"MEMBRANOUS WEB" FORMATIONA characteristic feature of plus-strand RNA viruses is their propensity to replicate their genome in close association with host intracellular membranes. These

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membrane platforms can either be pre-existing membrane organelles or membrane structures induced de novo by the virus (Chu and Westaway, 1992; Froshauer et al., 1988; Lazarus and Barzilai, 1974; Rice, 1996). Egger et al. (Egger et al., 2002) investigated by electron microscopy the capacity of HCV proteins to elicit such

intracellular membrane alterations by expressing HCV proteins individually or in the context of the entire HCV polyprotein. Expression of the latter was associated with the induction of a novel membrane structure designated "membranous web" which appeared to consist of vesicles within a membranous matrix. Expression of NS4B alone also induced the membranous web. The emergence of the latter appeared to coincide with a reduction in the rough endoplasmic reticulum (RER), and regions of continuity between the RER and membranous web were observed (Egger et al., 2002). This suggested that the membranous web was derived from the endoplasmic reticulum (ER). Similar structures have been described in livers of HCV-infected chimpanzees (Pfeifer et al., 1980). Immuno-EM experiments revealed that all examined HCV proteins could be found associated with the membranous

web, suggesting that these proteins might form a membrane-associated multi-protein complex. Membranous web structures were also found in cells harboring HCV replicons (Gosert et al., 2003). Importantly, viral plus-strand RNA could be localized to these sites using a digoxigenin-labeled riboprobe followed by a gold conjugated anti-digoxigenin antibody. Finally, nascent viral RNA synthesis detected by metabolic labeling with BrU in the presence of actinomycin D (Gosert et al., 2003) co-localized with immunofluorescently-detected NS5A. Similar apparent co-localization of viral RNA and NS proteins has been described by others (Egger et al., 2002; El-Hage and Luo, 2003; Shi et al., 2003). It was therefore postulated that the membranous web represents the candidate site for HCV replication.

It remains to be clarified whether the membranous web, the MAFs described earlier (Gretton et al., 2005), and the speckle-like structures detected by immunofluorescent probes against HCV RNA and proteins in cells harboring subgenomic replicons (Gosert et al., 2003) are all identical structures representing viral replication sites, or different structures with different functions. It will also be important to confirm the existence and character of membranous webs in cells which are capable of permitting all aspects of the viral life cycle (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005).

Nevertheless, NS4B's apparent key role in the establishment of the replication (and possible early assembly) platform represented by the membranous web places NS4B in a particularly prominent position in the viral life cycle. Moreover, because the membranous web is not a normal feature of host cells, selective inhibition of NS4B-induced membranous web formation could represent a specific antiviral strategy with low inherent cytotoxicity.

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MODULATION OF ER FUNCTIONSThe ER is a major subcellular organelle with which the HCV life cycle is associated. The HCV proteins are translated on, undergo initial processing by, and associate with the ER. Moreover, as mentioned above, the membranous web is postulated to be derived at least in part from the ER. It should therefore not be too surprising that HCV in turn can affect a variety of ER functions.

In poliovirus, two different virally-encoded proteins slow the rate of ER-to-Golgi traffic (Doedens and Kirkegaard, 1995) reducing the rate of ER protein secretion and imparing the presentation of major histocompatibility complex class I (MHC-I) antigens on the cell surface (Deitz et al., 2000). To determine whether HCV proteins might induce similar effects, changes in anterograde traffic from the ER to the Golgi apparatus were determined as a function of HCV NS protein expression (Konan et al., 2003). For this, they monitored the glycosylation status of coexpressed vesicular stomatitis virus G protein (VSV-G), a classical technique for secretory pathway trafficking studies (Lodish et al., 1983). As VSV-G is transported from the ER to the Golgi, the sensitivity of the covalently attached sugars to endo H digestion is altered, thus providing a convenient marker of anterograde trafficking. Of all the NS proteins evaluated, including putative partially-processed precursor proteins, only a fused NS4A/B affected the rate of ER-to-Golgi traffic, reducing it by approximately three fold. Interestingly, no effect was seen with the fully-processed products NS4A or NS4B alone, or in combination. NS4A/B expression inhibited the secretion of other cargo proteins as well. In cells harboring full length HCV replicons, MHC-I appearance on the cell surface was attenuated by three- to five fold compared to control cells. Both NS4A/B and NS4B caused the accumulation

of clustered, aggregated membranes in 293T cells and were found localized to these membranes. Only NS4A/B caused the formation of swollen vesicles, but the protein did not localize to these structures. These swollen vesicles were suggested to be ER-derived membranes swollen with cargo due to the blockage in ER-to-Golgi traffic. It was postulated that such blockage –with the associated reduction of cytokine secretion and transport of membrane proteins such as MHC-I to the cell surface--could affect the host immune response to HCV infection.

HCV has also been implicated in the induction of ER stress (for review see Tardif et al., 2005) and this may contribute as well to the decrease in MHC-I expression found in cells harboring HCV replicons (Tardif and Siddiqui, 2003). NS4B may play both a role in the induction of ER stress and its regulation. One mechanism may reside with the ATF6 (activating transcription factor 6) activation associated with HCV replication (Tardif et al., 2002). ATF6 is a transcription factor activated to alleviate ER stress when protein folding is disrupted. Using a yeast two-hybrid assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP), also called ATF6β, was identified to interact with NS4B (Tong et al., 2002). The

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N-terminal half of NS4B and a central portion of CREB-RP/ATF6β containing the basic leucine zipper (bZIP) domain were involved in this interaction. ATF6α, which shares high sequence similarity with CREB-RP/ATF6β, was also shown to interact with NS4B in yeast although the interaction was weaker than that between NS4B and CREB-RP/ATF6α. Interestingly, ATF6β suppresses transcription of ER stress-inducible genes while ATF6α enhances it (Thuerauf et al., 2004). This might suggest that NS4B can, like NS5A and E2 (Gale et al., 1997; Pavio et al., 2003; Taylor et al., 1999), inhibit specific downstream pathways of ER stress induction. At present, however, interactions of NS4B with ATF6 in vivo and the functional consequences remain to be determined. Finally, it is possible that some of the above effects are consequences of NS4B's interactions with membranes per se, including the diversion of ER components into the creation of the membranous web.

MALIGNANT TRANSFORMATIONThe leading cause of hepatocellular carcinoma (HCC) in the US is hepatitis C virus. Typically, this severe complication of HCV occurs many years after infection, in the setting of cirrhosis. Because the latter is an independent risk factor for HCC, HCV-associated HCC could either be simply an indirect consequence of HCV-induced cirrhosis. Alternatively, the HCC could be the direct result of specific viral factors, presumably in the context of a "multi-hit" scenario where the time course for full accumulation of these hits parallels the development of cirrhosis. In the context of the latter possibility, several HCV proteins including the core protein and non structural proteins NS3 and NS5A have been reported to transform various cell lines, and in the case of core cause tumors when expressed in transgenic mice (Moriya et al., 1998). The cell transformations occur either alone or in cooperation with other known oncogenes (Ghosh et al., 1999; Ray et al., 1996; Ray et al., 2000; Sakamuro et al., 1995). The involvement of HCV's NS protein NS4B in tumor formation was also investigated (Park et al., 2000). NIH3T3 cells co-transfected with NS4B and the Ha-Ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent growth--all characteristics of a transformed phenotype. Similar experiments using c-src, c-fos , c-myc substituted for the Ha-ras, failed to show any tumorigenic phenotypes, suggesting a specificity for enhancement of Ras-mediated pathways. Since many viral proteins are involved in Ras-mediated transcriptional regulation and growth control through AP1 activation, the effect of NS4B on luciferease activity controlled by the AP1 promoter was examined (Park et al., 2000). The luciferase gene was cloned under the control of the AP1 promoter and transfected into NIH3T3 cells stably co-transfected with NS4B and Ha-ras. Luciferase activity in these cells was increased by six fold in comparison with cells stably transfected with Ha-ras alone. AP1-Luc transfection into stable NS4B transfectants did not increase AP1-Luc activity. This suggests that the apparent synergy between NS4B and Ha-ras might be mediated via AP1 activation. Because of the limitations associated with interpreting experiments involving overexpression

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and in vitro transformation correlates, the relevance of the above (albeit provocative) observations to clinical HCV-associated HCC remains to be determined

NS4B FEATURES THAT MAY UNDERLIE THE MECHANISMS OF THE ABOVE FUNCTIONS

THE NS4B AMPHIPATHIC HELIXSimilar to NS5A (Elazar et al., 2003), NS4B has a predicted N-terminal amphipathic helix (see Fig. 2) which suggested another mechanism of membrane association in addition to NS4B's TMDs (Elazar et al., 2004). This amphipathic helix (AH) was found to be conserved across all HCV isolates, suggesting it plays a critical role in productive natural infections. Introduction of mutations designed to disrupt the hydrophobic face of the AH abolished its ability to mediate membrane association.

This disruption abolished HCV RNA replication, whereas mutations designed to only partially disrupt the amphipathic nature of the AH resulted in an intermediate level of replication (Elazar et al., 2004). These results genetically validate the NS4B AH as a potential antiviral target, although the mechanistic details underlying the NS4B AH's critical role in replication await further definition. One possibility may be to help mediate the establishment of the HCV replication complex.

Fig. 2. The N-terminus of NS4B harbors a predicted amphipathic helix. The amino-terminal segment of NS4B is predicted to adopt an alpha helical secondary structure, depicted here in a helix net diagram wherein the cylindrical alpha-helical segment is "sliced" longitudinally along one face and "flattened" into the plane of the page. Amino acids in the N- to C-terminal direction is shown beginning at proline 5. Hydrophobic amino acids are shaded in grey. Note the continuous stretch of such amino acids along one side of the helix, defining its amphipathic nature.

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When NS4B is expressed from a subgenomic replicon with a mutated NS4B AH, localization of NS4B is aberrant and the cytoplasmic speckle-like pattern typical of wild type replicon cells is lost (Elazar et al., 2004). The mutant NS4B retains a reticular staining pattern suggestive of ER localization, but it is unable to be further sublocalized into the characteristic speckles. Moreover, not only is normal NS4B localization abrogated, but the disrupted NS4B AH prevents other members of the HCV replication complex form coalescing into the speckled pattern associated with replication-competent replicons. Thus the NS4B AH may be responsible for mediating the association of NS4B and replication complex components with lipid rafts. The AH is also hypothesized to play a role in membranous web formation. Interestingly, a second AH has also been identified within NS4B (Glenn and Elazar, unpublished data), which may also play an important role in the viral life cycle.

NS4B HAS A NUCLEOTIDE BINDING MOTIFInspection of the NS4B primary sequence revealed the presence of a candidate nucleotide binding motif (NBM) beginning in the middle of the protein. Such NBMs are characterized by conserved of sets amino acids present in proteins known to bind nucleotides. The most conserved elements of NBMs are the so-called A motif (GxxxxGK) and B motif (DxxA) which are separated by a variable number of amino acids, depending on the particular protein (Gorbalenya and Koonin, 1989). Additional motifs common in a large number of GTP-binding proteins, such as the G-protein superfamily, can be identified. Among these are the G and PM2 motifs consisting of single amino acids (F and T, respectively) located between the A and B motifs (Fig. 3).

The crystal structures of several G-proteins has revealed that the G and PM2 elements interact with the nucleotide base (guanine in the case of G-proteins) and the chelated Mg++ ion, respectively (Stenmark and Olkkonen, 2001).

NS4B was found to specifically bind GTP (Einav et al., 2004). Similar to many other nucleotide-binding proteins, NS4B was also able to hydrolyze nucleotide, indicating it is a GTPase. Mutations disrupting the A motif element of the NBM impaired GTP binding and hydrolysis. These same mutations dramatically inhibited HCV RNA replication, and the effects on GTPase activity paralleled the effect on replication (Einav et al., 2004). Further mutagenesis experiments disrupting the B and the G motifs showed similar effects on viral replication (Moon and Glenn unpublished results). None of these mutations had any apparent effect on NS4B protein levels or its targeting to the ER. Together these results suggest that the nucleotide binding motif within NS4B is essential for mediating NS4B's role in HCV replication in vitro. The requirement of a nucleotide binding motif for productive viral infection in vivo is further suggested by the conservation of this motif across natural HCV isolates of all genotypes. Although it is clear that this NBM mediates

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critical functions in the viral life cycle the exact details of its function await further definition. One possibility can be that the NS4B NBM mediates binding of nucleotides not only as single molecules but also as part of a polynucleotide structure such as RNA. By simultaneously binding cellular membranes and RNA, NS4B might contribute to the structural integrity of the replication complex by helping to anchor it to membranes.

The ability to bind and hydrolyze GTP has evolved to serve diverse regulatory roles in biology, in part because it represents an efficient and regulateable molecular switch. As such, the NS4B NBM affords a wide variety of potential regulatory mechanisms and it can be readily envisaged to mediate many of the effects ascribed to NS4B in this chapter. Because the amino acids upstream and downstream of the NBM are highly conserved across HCV isolates, yet very different from known host cell G-proteins, there is also the potential for selective inhibition of the NS4B NBM.

FUTURE DIRECTIONSAs reviewed in the preceding sections, mounting evidence indicates the importance of NS4B to various viral activities. NS4B also appears to be connected with various viral effects on the host cell. It is quite clear that to mediate all these effects NS4B

Fig. 3. Elements of NS4B's nucleotide binding motif (NBM). The NS4B protein is depicted schematically with its 4 predicted TMDs in relation to the ER membrane. The relative positions and amino acid composition of the A motif, B motif, G and PM2 motifs are indicated (black)—together these elements constitute the NS4B NBM. Also noted is the position of the amphipathic helix (white). Numbers correspond to amino acid positions. See text for details.

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likely has a variety of cellular and/or viral protein partner(s). Uncovering their identity may further clarify some of NS4B's functions—many of which still have unproven mechanisms. Important information might be gained from investigating common features in the NS4B proteins of different viruses from the Flaviviridae family. For example, the related bovine viral diarrhea virus isolates divide into cytopathic and noncytopathic biotypes. In all noncytopathic biotypes that arouse from cytophatic variants an Y2441C substitution in NS4B was found. This might implicate the involvement of NS4B in viral cytopathogenicity (Qu et al., 2001).

Although information about NS4B is continuing to accumulate, several key points relating to its currently ascribed functions remain unclear. For example, only 2 of the four to six predicted TMDs in NS4B have experimental validation. Understanding the exact topology of NS4B could assist in further revealing some of its functions and in the design of specific inhibitors. Another issue awaiting further clarification is the exact intracellular localization of NS4B and its relationship to viral replication. The NS4B-induced membranous webs, MAFs, and the characteristic NS4B speckles may or may not be the same structures. Moreover, which of these represents the authentic sites of viral replication remains to be clarified. NS4B's inhibitory effects on the host translation machinery seem somewhat clear but its exact effect on viral IRES-mediated translation remains uncertain. Improving the understanding of these issues might provide the requisite tools to specifically control viral protein translation. Similarly, the critical role of NS4B's NBM in the viral life cycle has been demonstrated but the exact details of the function(s) mediated by the GTPase activity remain to be fully described. Although there are undoubtedly yet to be discovered features of NS4B, its already identified properties clearly make it a valuable probe of host cell biology.

Both the NS4B AH and NBM provide potential mechanisms to mediate many of the proposed functions for NS4B. The ability to pharmacologically inhibit these domains thus represents another exciting avenue for future research. With respect to the AH, similar strategies as those shown to be effective against the NS5A AH (Elazar et al., 2003) can be readily adapted to the NS4B AH target. The NBM may offer even more readily adaptable antiviral strategies.

Further characterization of the possible role of NS4B in malignant transformation may advance the understanding of HCV-associated carcinogenesis mechanisms and may lead to novel therapeutic strategies. Alternatively, effective pharmacologic eradication of HCV could by itself make the leading cause of hepatocellular carcinoma in the US theoretically preventable. By analogy with other infections, such as tuberculosis or HIV, this type of pharmacotherapy is most likely to consist of a cocktail which includes multiple agents, each designed against an independent virus-specific target. Exploitation of current and yet to be identified targets within

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NS4B could increase the repertoire of agents available for inclusion in such therapeutic cocktails of the future.

FUTURE DIRECTIONSThis work was supported by ROIDK066793 and Burroughs Wellcome Career Award (to JSG).

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Chapter 9

HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication

Yupeng He, Kirk A. Staschke and Seng-Lai Tan

ABSTRACTThe hepatitis C virus (HCV) non-structural 5A (NS5A) protein has generated wide interest in HCV research because of its ability to modulate the host cell interferon (IFN) response. The protein is phosphorylated on multiple sites by host cell kinases and interacts with host cell membranes. While no known enzymatic function has been ascribed to NS5A, it is an essential component of the HCV replicase and exerts a wide range of effects on cellular pathways and processes, including innate immunity and host cell growth and proliferation. In this chapter, we review the many studies describing the interaction of NS5A with viral and host cell proteins, its ability to modulate multiple cellular pathways, and its recently described structural attributes, subcellular localization, and function during HCV replication.

INTRODUCTIONTranslation of the HCV genome results in the production of a large polyprotein, from which NS5A is processed by the NS3 protease (Reed and Rice, 2000). As a nonstructural (NS) protein with no apparent enzymatic activity, NS5A functions through interaction with other viral and cellular proteins. Its primary amino acid (a.a.) sequence predicts a proline-rich, predominantly hydrophilic protein with no obvious trans-membrane helices. NS5A exists as multiple phospho-isoforms and is predominantly localized in the cytoplasmic/perinuclear compartments of the cell, including the ER and the Golgi apparatus. This pattern of NS5A localization is consistent with the notion that NS5A interacts with multiple host cell and viral proteins. There is strong evidence that NS5A is also localized in certain modified cytoplasmic membrane structures during HCV replication, where it plays a functionally significant role as part of the HCV replication complex or replicase. NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions, and viral pathogenesis.

STRUCTURAL FEATURES AND SUBCELLULAR LOCALIZATION OF NS5AEarly studies utilizing cells in which NS5A had been overexpressed or liver biopsy samples from chronic HCV patients, showed that NS5A is localized in the

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cytoplasm and the perinuclear membrane fraction, consistent with localization to the ER/Golgi (Ide et al., 1996; Polyak et al., 1999; Tanji et al., 1995a). In addition, when expressed either alone or in the context of additional HCV NS proteins (an NS3-5B polyprotein) in human hepatoma cells, NS5A was also found to co-localize with the HCV core protein on the surface of globular structures containing lipid droplets (Shi et al., 2002). Interestingly, previous studies had noted that NS5A binds to the core protein on membrane structures (Goh et al., 2001). Also, NS5A was found to bind to Apolipoprotein A1 (ApoA1), a protein component of high-density lipoprotein (HDL) particles and co-localized with ApoA1 in the Golgi (Shi et al., 2002). In other studies, NS5A was found to bind to a snare-like protein called hVAP-33 (Tu et al., 1999). In HCV replicon cells, NS5A bound to hVAP-33 and localized to detergent-resistant lipid rafts (Gao et al., 2004). In a study utilizing an HCV replicon in which NS5A was fused to green fluorescent protein (GFP), the NS5A-GFP fusion protein was associated with brightly fluorescent dot-like structures in the cytoplasm (Moradpour et al., 2004). Analysis of these structures by electron microscopy led to their description as "membranous webs". It was suggested that these might represent sites of bound replication complexes or sites of virus assembly since HCV NS proteins and nascent viral RNA all co-localized to these structures (Moradpour et al., 2004). Given its interaction with host cell proteins and membranes, as well as the HCV core protein (Goh et al., 2001), it is possible that NS5A may also regulate HCV virus assembly directly through its interaction with the viral capsid protein. So it seems that after its expression and processing in the ER, the NS5A protein localizes into specialized cytoplasmic membrane structures, as a part of putative HCV replication and/or assembly complexes. These specialized membrane structures may be derived from or related to either ER or Golgi, and the association of NS5A with these structures may require interaction with host-derived, membrane-associated proteins or other viral proteins. The importance of cellular membranes is underscored by the recent finding that inhibitors of protein geranylgeranylation (Ye et al., 2003) and fatty acid biosynthesis (Kapadia, 2005) block HCV replication in replicon cells. Recently, FBL-2, a geranylgeranylated cellular protein was shown to bind to NS5A and found to be critical for HCV replication (Wang et al., 2005). Whether or not additional cellular proteins play a role in these processes is not known.

The membrane association of NS5A protein occurs post-translationally and NS5A has similar properties to that of an integral membrane protein (Brass et al., 2002). A membrane-anchoring region was mapped to the N-terminal 30 a.a. of NS5A which form a highly conserved amphipathic α-helix (Brass et al., 2002). It was suggested that membrane anchorage is mediated by the hydrophobic side of the amphipathic helix, resulting in an orientation parallel to the lipid bilayer, while positioning the helix in the cytoplasmic leaflet of the ER membrane. A follow-up study demonstrated that the N-terminal amphipathic helix is not only necessary and sufficient for

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membrane localization, but also important for HCV replication since mutations disrupting helix formation impaired HCV replication (Elazar et al., 2003). A three-dimensional structure of this region was solved by NMR spectroscopy (Penin et al., 2004). The structure revealed an α-helix extending from a.a. 5 to 25. The helix contains a hydrophobic side embedded in detergent micelles, and a solvent-exposed, polar, charged side. Confirmatory studies showed that the NS5A membrane anchor region forms an in-plate, amphipathic α-helix, embedded in the cytosolic leaflet of the membrane bilayer. It was also suggested that this region is not only involved in membrane localization, but also required for additional functions, as mutations of conserved residues on the cytosolic face impaired HCV replicon RNA replication without affecting membrane association (Penin et al., 2004). In addition to its role in localizing NS5A protein to appropriate membrane compartments, it is likely that the membrane anchor region provides a platform for protein-protein interactions involved in the HCV replication process. It is also possible that specific cytosolic residues of the helix may contribute to protein-protein interactions with additional viral and cellular components.

Until recently, structural information on the NS5A protein had been limited, largely due to the difficulty in purifying the full-length protein. Early studies on NS5A structure were limited to individual structural motifs and their functions. With the recent characterization of its domain organization and resolution of a structure of the N-terminal region (Tellinghuisen et al., 2004; Tellinghuisen et al., 2005), we can begin to gain an appreciation for the multi-dimensional structure of the NS5A protein. A recent study using bioinformatics-assisted modeling suggested a three-domain organization (Tellinghuisen et al., 2004) with domain I (a.a. 1-213) located in the N-terminal region, and Domain II (a.a. 250-342) and Domain III (a.a. 356-447) in the C-terminal region (Fig. 1). This organization was confirmed by limited proteolysis experiments. Interestingly, an unconventional zinc-binding motif was predicted to exist in the N-terminal domain, indicating that NS5A is a zinc metalloprotein (Tellinghuisen et al., 2004). The predicted zinc-binding motif involves four cysteine residues (C39, C57, C59, and C80; Fig. 1), and includes a structural motif (CX17CXCX20C) that is well conserved among Hepaciviruses and Pestiviruses. In this same study, the zinc content of purified NS5A protein or the N-terminal domain alone was determined and it was found that each protein molecule coordinates one zinc atom. This motif appeared critical for the structural stability and function of the NS5A protein, since mutation of any single cysteine residue in the motif disrupted the ability of NS5A to coordinate zinc and eliminated HCV replicon RNA replication (Tellinghuisen et al., 2004). A more recent study reported the crystal structure of NS5A Domain I (a.a. 36-198) at 2.5-A resolution (Tellinghuisen et al., 2005). The structure revealed the presence of a novel fold, a zinc-coordination motif, and a C-terminal disulfide bond. Mutational analysis suggested that the disulfide bond is not required for the HCV replicase functions of

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NS5A (Tellinghuisen et al., 2004). These studies have provided a nice starting point for understanding the structural organization of NS5A, the elucidation of structural assembly points of NS5A as it pertains to its role as an HCV replicase subunit, and NS5A's ability to interact with multiple host cell proteins and molecules.

While most studies have focused on the membrane associated forms of NS5A, an early study identified a putative nuclear localization signal (NLS) sequence (PPRKKRTVV; a.a. 354-362) within the C-terminal half of NS5A (Ide et al., 1996) (Fig. 1). This sequence appeared to function as an NLS since it was able to target a heterologous protein (β-Galactosidase of E. coli) into the nucleus. The presence of an NLS suggests a possible nuclear localization and function of NS5A in addition to its membrane bound isoforms. One study suggested that the localization of NS5A to membranes is at least partially determined by its most N-

Fig. 1. Schematic diagram of the NS5A protein. Several prominent features of the NS5A protein and described in detail in this review are shown. The three domain structure of NS5A (Tellinghuisen et al., 2005) is depicted. The N-terminal amphipathic α-helix (Elazar et al., 2003; Penin et al., 2004), the IFN sensitivity determining region (ISDR) (Tan and Katze, 2001), the class I and class II proline-rich (Tan et al., 1999), and NLS sequences (Ide et al., 1996) are also shown. In addition, basal and hyperphosphorylation sites as well as the binding sites for several interacting proteins (see Table 1) are noted.

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terminal region (Satoh et al., 2000). It was found that NS5A mutants lacking this region were localized in the nucleus. Conversely, the N-terminal 27 a.a. from NS5A were capable of retaining a nuclear protein in the cytoplasm. In addition, a cleaved form of NS5A protein missing the N-terminal region (a.a. 155-389) also localized to the nucleus. The N-terminal sequence was able to block the function of the NLS in the C-terminal region and prevented NS5A protein from being transported into the nucleus (Song et al., 2000). This putative "NLS-masking-sequence" in the N-terminus, which appears to overlap with the amphipathic α-helical region, did not function as a nuclear export signal. So it seems that the this region can also regulate the function of the NLS and thus the nuclear localization of NS5A protein, presumably by preferentially targeting NS5A protein into cytoplasmic membrane structures. It is likely that the localization of NS5A protein in different subcellular compartments is determined and regulated by different structural features and/or different forms of the protein, and the differential localization of NS5A in different compartments may contribute to its different biological functions. In particular, the cytoplasmic vs. nuclear localization and function of NS5A could be carefully counter-regulated and balanced through its different structural motifs regulating subcellular localization of the protein during the viral life cycle. Along these lines, the C-terminal half of NS5A contains a positively charged region enriched with acidic and proline residues, a structural feature resembling those of eukaryotic transcriptional activators (Chung et al., 1997; Ide et al., 1996). Following deletion of the N-terminal membrane anchoring domain, the C-terminal half of NS5A functioned as a potent transcriptional activator when fused to the DNA-binding domain of yeast GAL4 protein, in both yeast and human hepatoma cells (Chung et al., 1997; Kato et al., 1997; Tanimoto et al., 1997). Furthermore, a region between a.a. 130-352 was found to be critical for optimal transcriptional activation (Tanimoto et al., 1997). These studies suggest that truncated forms of NS5A may localize to the nucleus via the cryptic NLS only after removal of the N-terminal membrane-anchoring region and regulate cellular gene transcription. The mechanism of NS5A nuclear localization may involve proteolytic processing of NS5A. Indeed, this was observed and a cleaved form of the protein was able to localize to the nucleus and caused transcriptional activation when the alpha subunit of PKA was co-expressed (Satoh et al., 2000; Song et al., 2000). The NS5A cleavage in mammalian cells was enhanced by apoptotic stimuli and was inhibited by the caspase inhibitor Z-VAD-FMK, suggesting that a caspase-like protease(s) contributes to the cleavage of NS5A (Satoh et al., 2000). A later study showed that NS5A protein was also cleaved following induction of apoptosis by the HCV core protein and that the proteolytic processing of NS5A could be inhibited by Z-VAD-FMK (Goh et al., 2001). These studies indicated that NS5A protein cleavage is likely mediated by caspase(s) and/or related protease(s) and may be linked to the induction of apoptosis. In support of this, a recent study found that NS5A was processed into multiple forms in different mammalian cell types (Vero, HepG2, Huh-7, and WRL68), and suggested that both

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caspase-like proteases and calcium-dependent calpain proteases were involved in NS5A processing (Kalamvoki and Mavromara, 2004). However, this study also showed that both the cleaved and full-length forms of NS5A exhibited a cytoplasmic/perinuclear localization. Although these results suggest that additional proteolytic processing of NS5A may occur, the biological function the cleaved forms in the context of HCV biology remain uncertain at the moment.

NS5A PHOSPHORYLATION: A FUNCTIONAL ROLE OR RED HERRING?Studies on NS5A expressed in tissue culture revealed predominantly two forms of NS5A protein with differing apparent molecular weights of 56 and 58 kDa. Basal phosphorylation results in expression of the 56 kDa isoform while hyperphosphorylation results in the 58 kDa form(Kaneko et al., 1994; Tanji et al., 1995b). In addition, there is evidence that p58 is converted from p56 and requires polyprotein processing (Neddermann et al., 1999). Phosphorylation of NS5A protein occurs predominantly on serine residues, with a minor fraction on threonine residues (Kaneko et al., 1994; Reed et al., 1997; Tanji et al., 1995b). A number of serine residues (2194, 2197, 2201, and/or 2204) in the central region of NS5A were found to be important for hyper-phosphorylation, and two other regions (a.a. 2200-2250 and the C-terminal region) appeared important for basal-phosphorylation (Tanji et al., 1995b) (Fig. 1). In addition, a major phosphorylation site was identified as serine 2321, which is located within the C-terminal Class II proline-motifs and likely represents a basal phosphorylation site (Reed and Rice, 1999). In another study, Katze and colleagues identified the major phosphorylated residue on an NS5A phosphopeptide (a.a. 2193-2212) as serine 2194, which is well conserved among HCV genotypes and presumably is a site for hyper-phosphorylation (Katze et al., 2000). Additional phosphorylation sites remain to be mapped. In addition, phosphorylation of NS5A on tyrosine has not been reported. Interestingly, NS5A proteins from other viruses closely related to HCV, such as BVDV and YFV, were also phosphorylated in various in vitro and in vivo systems, and it appeared that phosphorylation occurred via serine/threonine kinase(s) (Reed et al., 1998). These results indicate that NS5A phosphorylation is a well-conserved feature, and either the phosphorylation of NS5A itself or NS5A interaction with its cellular kinases plays an important role in the Flavivirus life cycle. Whether these NS5A proteins from the different virus species are phosphorylated by the same or related kinase(s) is completely unknown. However, one study reported that NS5A protein from HCV genotype-2a was not hyperphosphorylated in contrast to that of NS5A from genotype-1 (Hirota et al., 1999). This raises the question as to whether the same phosphorylation pattern is prevalent throughout all HCV genotypes/isolates, and whether different phosphorylated forms of the NS5A protein play different roles in viral pathogenesis or in the HCV viral life cycle. It is also possible that the different NS5A phosphorylation patterns in vitro were caused by differences in

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the experimental systems employed. Unfortunately, due to technical limitations, the phosphorylation of NS5A in the liver of chronic HCV patients is not easily addressed.

Numerous studies have attempted to identify the cellular kinase(s) responsible for NS5A phosphorylation. NS5A protein was found to stably associate with an unknown protein kinase(s) from mammalian cells, and this kinase was able to phosphorylate native NS5A protein on serine residues in vitro (Ide et al., 1997). This same study also showed that the catalytic subunit of cAMP-dependent protein kinase A (PKA) was capable of phosphorylating NS5A in vitro. Interestingly, as previously noted, co-expression of the alpha subunit of PKA seemed to affect the transcriptional activity of a cleaved form of NS5A (Satoh et al., 2000). However, there is no evidence that PKA is an NS5A kinase in mammalian cells. By testing the effect of various kinase inhibitors on NS5A phosphorylation in vitro and examining the context of known phosphorylation sites, other studies suggested that the NS5A kinase(s) belongs to the CMGC group of serine-threonine kinases and is likely a proline-directed kinase (Katze et al., 2000; Reed and Rice, 1999; Reed et al., 1997). Indeed, casein kinase II (CK II), a member of the CMGC kinase family, was found to phosphorylate NS5A protein in vitro, and it showed the same molecular size and properties as an unknown kinase that stably associates with NS5A in mammalian cells through the N-terminal region of NS5A (Kim et al., 1999). Thus, CK II stands as a candidate kinase for NS5A phosphorylation, but direct evidence for its role in vivo remains elusive. A more systematic approach was employed by Coito and colleagues, who performed a global screening of all yeast kinases capable of phosphorylating NS5A in vitro, and then attempted to predict and identify homologous mammalian kinases that were also capable of phosphorylating NS5A through both bioinformatic and biochemical methods (Coito et al., 2004). By comparing in vivo and in vitro NS5A phosphopeptide profiles, their results suggested that several mammalian kinases (AKT, p70S6K, MEK1, and MKK6) might be responsible for NS5A phosphorylation in vivo. In particular, the functional relevance of p70S6K or related kinases was further supported by the fact that rapamycin was able to reduce the phosphorylation of specific NS5A phosphopeptides in vivo. Given the complexity of NS5A phosphorylation, it is likely that multiple kinases are involved and that phosphorylation occurs in a regulated and coordinated manner.

Several lines of evidence suggest that the pattern NS5A phosphorylation is dependent on additional HCV NS proteins. One group reported that the level of the hyperphosphorylated form of NS5A (p58), was enhanced by the presence of NS4A. Additionally, the association of NS5A with NS4A through a.a. 2135-2139 of NS5A was important for NS4A-dependent phosphorylation (Asabe et al., 1997; Kaneko et al., 1994). A later study suggested that the appearance of p58 required

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NS2 in cis and the autoproteolytic activity of the NS2-3 protease. The loss of p58 by disruption of NS2-3 autoproteolysis was rescued by expressing an NS2-3 in trans (Liu et al., 1999). However, other studies published at the same time showed that the presence of NS3-4A-4B in cis was necessary and sufficient for the hyperphosphorylation of NS5A (Koch and Bartenschlager, 1999; Neddermann et al., 1999) and the presence of NS3-4A protease activity in cis was absolutely required for p58 production (Neddermann et al., 1999). Interestingly, it was also found that single a.a. mutations with NS3, as well as mutations within NS4A and NS4B that do not disrupt polyprotein processing, also affected NS5A hyperphosphorylation (Koch and Bartenschlager, 1999). In summary, the exact requirement for other HCV NS proteins and their roles in NS5A phosphorylation is not completely understood, but it seems likely that NS5A phosphorylation is regulated in the context of other NS proteins, and requires both polyprotein processing and interactions among the NS proteins within a multi-subunit protein complex. Despite these observations, only recently has the role of NS5A phosphorylation been described in the context of HCV replication (Evans et al., 2004). These latter studies suggest that the differential phosphorylation of NS5A regulates its function during HCV replication, presumably by affecting its interaction and formation of protein complexes with other proteins.

EMERGING ROLE OF NS5A IN HCV REPLICATIONStudies utilizing subgenomic HCV replicons in cell culture systems suggest that NS5A plays an important role in the establishment of high-level HCV RNA replication (see Chapter 11). Several adaptive mutations that confer higher replication efficiency to HCV replicons are clustered in the NS5A region, and some of these adaptive mutation sites either overlap with putative NS5A phosphorylation sites or have been shown to affect NS5A hyperphosphorylation. This re-opened the question as to whether NS5A phosphorylation plays a role in HCV replication. Interestingly, when expressed alone in mammalian cells in culture, NS5A has an apparent half-life of four to six hours (Polyak et al., 1999). In replicon cells, the hyperphosphorylated (p58) form of NS5A is much less stable than the basally phosphorylated (p56) form of the protein (Pietschmann et al., 2001), suggesting possible differences in function. Indeed, one study found an inverse relationship between NS5A phosphorylation level and its interaction with hVAP-33, which in turn, is required for HCV replication in the replicon system (Evans et al., 2004). In addition, some of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and increased NS5A binding to hVAP-33. It is noteworthy that the region of NS5A that interacts with hVAP-33 encompasses the putative NS5A hyperphosphorylation sites (Fig. 1). NS5A also binds directly to the NS5B viral polymerase both in vitro and in vivo (Shirota et al., 2002). This interaction was suggested to modulate the enzymatic activity of NS5B (Shirota et al., 2002) and in replicon cells shown to be critical for HCV replication (Shimakami et al., 2004). A

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model has been proposed in which the phosphorylation status of NS5A serves as a molecular switch in the regulatory process of HCV RNA replication by affecting the association between NS5A and other components of the viral replication complex (Evans et al., 2004). This model also implies that host cell kinases regulate the HCV replication process through differential NS5A phosphorylation. In line with this working model, another study identified three undisclosed kinase inhibitors that blocked NS5A hyperphosphorylation in cell culture, and showed that treatment with any of these compounds stimulated replication of a wild-type replicon construct that has no adaptive mutations and replicates poorly otherwise (Neddermann et al., 2004). This is an exciting finding since this approach might allow efficient replication of many HCV strains that otherwise replicate very poorly in cell culture. This method may also open a way to establish different HCV replicon strains without the introduction of adaptive mutations. Thus, identifying the physiologically relevant NS5A kinases (and phosphatases) remains a high priority. Another prediction from the above working model is that p58, the hyperphosphorylated form of NS5A, is specifically linked to down-regulation of HCV RNA replication in cell culture. This prediction is further supported by results from a recent study, in which extensive mutagenesis analysis was carried out on a region of NS5A presumably involved in basal- and hyperphosphorylation (Appel et al., 2005). It was found that mutations in the central serine cluster reduced NS5A hyperphosphorylation and increased HCV replication. On the other hand, mutations of the C-terminal serine residues decreased the formation of p56, but did not affect HCV RNA replication significantly. Another study showed that the expression of a wild-type NS5A protein, or the introduction of a wild-type NS5A replicon in trans inhibited replication of NS5A-adapted replicons, in a dominant-negative fashion (Graziani and Paonessa, 2004). These results indicate that hyperphosphorylated wild-type NS5A may compete with the adapted-NS5A protein and down-regulate HCV RNA replication.

Despite these exciting results from HCV replicon-based studies, it has been shown that the adaptive mutations, especially those negatively affecting NS5A hyperphosphorylation, inhibit HCV replication following infection of chimpanzees (Bukh et al., 2002). In addition, similar adaptive mutations have not been observed in HCV patients. In fact, the putative hyperphosphorylation sites of NS5A are well conserved among different HCV genotypes/isolates from patients. These observations raised the concern over the physiological relevance of adaptive mutations in the HCV replicon system. In addition, questions as to whether the hyper-phosphorylation of NS5A serves additional biological roles during HCV infection in vivo have arisen. We may speculate that the hyper-phosphorylation of NS5A serves as a switch point between HCV RNA replication and downstream steps, such as virus capsid/particle assembly or virus particle maturation and release and that the hyperphosphorylated form of NS5A may be actively involved in these downstream events. As previously mentioned, an interaction between NS5A and the

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core protein has been noted (Goh et al., 2001; Shi et al., 2002). This model suggests that the basal- and hyper-phosphorylation of NS5A are regulated in a temporal fashion, presumably by different cellular kinases at different steps of the viral life cycle, to facilitate a complete, productive infection in vivo. With the recently establishment of bona fide HCV infection system in cell culture (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) (see Chapter 16), now it is possible to examine the differential phosphorylation status of NS5A and its role during different steps of the HCV infection cycle. Additional modes by which NS5A might affect HCV replication have also been suggested. Most recently, NS5A was shown to bind with high affinity to the 3' ends of HCV plus- and minus-strand RNAs (Huang et al., 2005). NS5A might also indirectly regulate HCV replication by modulating HCV IRES-dependent translation (He et al., 2003; Kalliampakou et al., 2005; Wang et al., 2003) and its ability to modulate cellular antiviral pathways stimulated by IFNs has been well documented (Gale and Foy, 2005; Tan and Katze, 2001).

NS5A AS A VIRAL INTERCEPTOR OF CELLULAR PATHWAYSInteraction with and modulation of host cell signaling pathways constitute an important aspect of many viral life cycles. HCV is no exception to this, and NS5A in particular may play a pivotal role in the interaction between HCV and cellular signal transduction pathways. The interplay between NS5A and the IFN system as well as the role of NS5A in IFN resistance has generated intense interest and has been extensively studied (Gale and Foy, 2005; Tan and Katze, 2001). In this section, we will focus on the affects of NS5A on additional cellular signaling pathways including those involved in growth, cell-cycle control, apoptosis and cell survival, and cellular stress responses.

NS5A has been shown to interact with a wide variety of host cell proteins and thus may modulate numerous diverse signal transduction pathways (Table 1). Among the cellular signaling pathways affected by the NS5A protein, the best characterized are those relating to cell proliferation and cell-cycle control, apoptosis and cell survival, and cellular stress responses. Despite many interesting results and insightful working models, in only a few cases has the functional relevance in the context of HCV replication been addressed. Thus, in most cases, the observations discussed below need further verification in model systems that can better simulate the HCV life cycle in vivo.

NS5A contains proline-rich PXXP motifs representing binding sites for SH3-domain containing proteins (Fig. 1). These motifs are frequently present in cellular signaling molecules (Tan et al., 1999). By testing a panel of SH3 domain-containing cellular proteins, Tan and colleagues found that NS5A specifically interacted with Grb2, a cellular adaptor protein involved in the growth factor signaling. The interaction of Grb2 with NS5A occurred through the C-terminal PXXP motif of

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NS5A (He et al., 2002; Tan et al., 1999). This interaction seemed to be mediated by the two SH3 domains of Grb2 in a cooperative fashion. Consistent with these findings, EGF stimulation of cells expressing NS5A showed reduced ERK and p38 MAPK activation, which are downstream signaling events mediated by the Grb2 adaptor protein. In addition, NS5A containing mutations within the C-terminal proline-rich motif neither interacted with Grb2, nor blocked EGF-stimulated ERK phosphorylation, supporting the direct connection between NS5A interaction with Grb2 and its effect on downstream MAPK pathways. The NS5A-Grb2 interaction and the inhibition of ERK phosphorylation by NS5A were also shown by another group in various mammalian cell types infected with recombinant HSV-1 viruses carrying NS5A (Georgopoulou et al., 2003). These studies suggest that NS5A can disrupt the MAPK mitogenic pathway through direct interaction with Grb2, either by preventing the recruitment of Grb2 to the upstream receptor complexes, or by disrupting Grb2 interaction with downstream components of the pathway, such as Sos. However, the original study found no evidence that NS5A reduced Grb2-Sos association. More recent studies have found that in HCV replicon cells there was reduced EGF receptor tyrosine phosphorylation and aberrant recruitment of the Shc and Grb2 adaptor proteins to the receptor. This correlated with reduced Shc phosphorylation and Ras activation (Macdonald et al., 2005a). While it is unclear whether the effects observed in replicon cells were caused by NS5A expression, it suggests that NS5A may disrupt the association of Grb2 and other adaptor proteins with the upstream receptor complex, thus blocking downstream Ras-Raf-MAPK activation at a very early step. The interaction between NS5A and Grb2, and the precise mechanism by which it blocks the downstream pathway need to be characterized in greater detail.

Grb2 and the downstream MAPK signaling pathways regulate many cellular processes such proliferation, gene expression, translational control, to name just a few. Thus targeting Grb2 and its downstream effectors through NS5A may have a significant influence on cellular functions and the HCV life cycle. In a follow-up study, it was found that NS5A inhibited the activity of AP1, a mitogenic and stress-activated transcription factor, through inhibition of the ERK pathway, and these effects were dependent upon the C-terminal Class II proline-rich motif that interacts with Grb2 (Macdonald et al., 2003). It was later shown in another study that an HCV replicon carrying a mutation within the C-terminal proline-rich motif lost the ability to block AP1 activation (Macdonald et al., 2005b). These results suggest that NS5A interaction with Grb2 may affect activation of the MAPK-dependent transcription factors and thus cellular gene expression. In addition, the ERK and p38 MAPK pathways also play a role in IFN signaling, by mediating serine phosphorylation of STAT1/3 transcription factors and contributing to maximal induction of IFN stimulated genes (He and Katze, 2002). Thus, in addition to its ability to modulate IFN responses directly by inhibiting the function of PKR, the ability of NS5A to

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Tabl

e 1.

Pro

tein

s rep

orte

d to

inte

ract

/ass

ocia

te w

ith N

S5A

.Pr

otei

n Pr

otei

n ca

tego

ryIn

tera

ctin

g N

S5A

regi

on(s

)In

viv

o in

tera

ctio

n?Bi

olog

ical

effe

ct/fu

nctio

n of

in

tera

ctio

nR

efer

ence

PKR

Cel

lula

r ant

ivira

l kin

ase

PKR

-BD

(a.a

. 237

-302

)Ye

sR

epre

ssio

n of

PK

R a

nd

dow

nstre

am p

athw

ays

Gal

e et

al.,

199

7; G

ale

et

al.,

1998

CK

IIC

ellu

lar C

MC

G fa

mily

ki

nase

N

-term

inal

regi

on?

-N

S5A

pho

spho

ryla

tion?

Kim

et a

l., 1

999

Grb

2C

ell s

igna

ling

adap

tor

prot

ein

C-te

rmin

al C

lass

II

prol

ine-

rich

mot

if Ye

sD

isru

ptio

n of

dow

nstre

am

mito

geni

c si

gnal

ing

Tan

et a

l., 1

999;

He

et a

l.,

2002

; Geo

rgop

oulo

u et

al.,

20

03; M

acD

onal

d et

al.,

20

03hV

AP-

33/h

VAP-

AC

ellu

lar m

embr

ane

prot

ein

a.a.

217

7-22

28Ye

sH

CV

RN

A re

plic

atio

n co

mpl

ex?

Tu e

t al.,

199

9; E

vans

et

al.,

2004

SRC

AP

Cel

lula

r tra

nscr

iptio

n fa

ctor

?

Yes

Dow

n-re

gula

tion

of p

21

prom

oter

act

ivity

Gho

sh e

t al.,

200

0

kary

ophe

rin b

eta

3?

?Ye

s?

Chu

ng e

t al.,

200

0C

dk1

Cel

l cyc

le c

ontro

l pro

tein

?Ye

sC

ell g

row

th a

nd c

ell c

ycle

pe

rturb

atio

ns?

Arim

a et

al.,

200

0

Cor

eH

CV

cap

sid

stru

ctur

al

prot

ein

a.a.

236

-354

Yes

HC

V v

irus p

artic

le a

ssem

bly?

Goh

et a

l., 2

001

p53

Cel

lula

r tra

nscr

iptio

n fa

ctor

?

Yes

Inhi

bitio

n of

tran

scrip

tiona

l tra

nsac

tivat

ion

by p

53

Maj

umde

r et a

l., 2

001;

La

n et

al.,

200

2; Q

adri

et

al.,

2002

Apo

A1

Cel

lula

r apo

lipop

rote

ina.

a. 1

-224

Yes

Col

ocal

izat

ion

in th

e G

olgi

ap

para

tus?

Shi e

t al.,

200

2

hTA

F(II

)32/

(TFI

ID)

Tran

scrip

tiona

l co

activ

ator

?Ye

sIn

hibi

tion

of tr

ansc

riptio

nal

trans

activ

atio

n by

p53

La

n et

al.,

200

2

NS5

BH

CV

pol

ymer

ase

a.a.

105

-162

and

277

-334

?R

egul

atio

n of

HC

V re

plic

atio

n?Sh

irota

et a

l., 2

002;

Sh

imak

ami e

t al.,

200

4p8

5 PI

3KC

ellu

lar k

inas

eN

-term

inal

regi

on; a

.a.

271-

300

Yes

Up-

regu

latio

n of

PI3

K/A

KT

surv

ival

pat

hway

He

et a

l., 2

002;

Stre

et e

t al

., 20

04

279

HCV NS5A

Gab

1C

ell s

igna

ling

scaf

fold

pr

otei

n-

Yes

Indi

rect

ass

ocia

tion

via

p85

PI3K

He

et a

l., 2

002

TBP

Cel

lula

r tra

nscr

iptio

n fa

ctor

In

dire

ct a

ssoc

iatio

n vi

a p5

3?Ye

sIn

hibi

tion

of tr

ansc

riptio

nal

trans

activ

atio

n by

p53

Q

adri

et a

l., 2

002

TRA

DD

Apo

ptos

is si

gnal

ing

adap

tor

Indi

rect

ass

ocia

tion

via

TRA

F2?

Yes

Pertu

rbat

ion

of T

RA

DD

si

gnal

ing/

apop

tosi

sM

ajum

der e

t al.,

200

2

TRA

F2C

ell s

igna

ling

adap

tor

prot

ein

Mid

dle

one-

third

(a.a

. 14

8-30

1)

Yes

Pertu

rbat

ion

of T

FAF2

sign

alin

g (N

FkB

, JN

K)

Park

et a

l., 2

002;

Par

k et

al

., 20

03am

phip

hysi

n II

Cel

lula

r ada

ptor

pro

tein

C-te

rmin

al p

rolin

e-ric

h re

gion

Yes

?Ze

ch e

t al.,

200

3

Bax

Cel

lula

r pro

-apo

ptot

ic

prot

ein

Bcl

-2 h

omol

ogy

dom

ains

Ye

sIn

hibi

tion

of a

popt

osis

Chu

ng e

t al.,

200

3

LaC

ellu

lar R

NA

-bin

ding

pr

otei

nN

-term

inal

regi

on (a

.a.

1-83

)?

?H

oush

man

d et

al.,

200

3

PTX

1C

ellu

lar h

omeo

dom

ain

prot

ein

?Ye

sPe

rturb

atio

n of

IFN

resp

onse

?G

hosh

et a

l., 2

003

2-5O

AS

IFN

indu

ced

antiv

iral

prot

ein

N-te

rmin

al (

a.a.

1-1

48)

Yes

Pertu

rbat

ion

of IF

N a

ntiv

iral

resp

onse

Tagu

chi e

t al.,

200

4

Hck

, Lck

, Lyn

, Fyn

Cel

lula

r Src

fam

ily

kina

ses

C-te

rmin

al C

lass

II

prol

ine-

rich

mot

if Ye

s?

Mac

Don

ald

et a

l., 2

004

HSP

27C

ellu

lar h

eat s

hock

re

sons

e pr

otei

nN

-term

inal

regi

on (a

.a.

1-18

1)Ye

s?

Cho

i et a

l., 2

004

Jak1

Cel

lula

r IFN

sign

alin

g ki

nase

?Ye

sST

AT3

activ

atio

nSa

rcar

et a

l., 2

004

FBL2

Cel

lula

r ge

rany

lger

anyl

ated

pr

otei

n

?Ye

sH

CV

repl

icat

ion

Wan

g et

al.,

200

5

He et al.

280

modulate MAPK signaling may also contribute to the ability of HCV to modulate the IFN response. In addition, NS5A also blocked phosphorylation of eIF4E following EGF stimulation, which may provide a mechanism for down-regulation of eIF4E-dependent translation of capped cellular mRNAs, thus favoring cap-independent translation of HCV RNA (He et al., 2001).

In addition to Grb2, the C-terminal proline-rich motif of NS5A was also found to mediate interaction with the SH3 domain of several Src kinase family members, including Hck, Lck, Lyn, and Fyn (Macdonald et al., 2004). NS5A interacted with these Src family members in vivo and differentially regulated their kinase activity, inhibiting Hck, Lck, and Lyn while activating Fyn. Similar findings were noted in HCV replicon cells. However, the downstream effects as well as the physiological role of the NS5A interaction with these Src kinases remain unclear. It seems quite remarkable that one particular motif of NS5A is able to interact with so many cell signaling molecules. Despite all these interesting findings, the physiological role of the NS5A C-terminal proline-rich motif remains unknown, since mutation of this motif in an HCV replicon did not affect HCV RNA replication (Macdonald et al., 2005a). It may be possible that the conserved proline-rich motif is involved in other steps of the HCV infection life cycle, and this issue might be addressed with the recently development HCV infection system in cell culture. Alternatively, this motif and its interaction with cellular proteins might be required for successful HCV infection and viral pathogenesis in patients, but not for HCV life cycle in tissue culture, in which many aspects of the in vivo infection are missing.

NS5A has also been shown to interact with and modulate another pivotal cellular pathway, the PI3K-AKT cell survival pathway (Macdonald et al., 2005a). NS5A directly interacts with the p85 regulatory subunit of PI3K through the SH3 domain of p85. This interaction may involve either the N-terminal region, or a novel motif within the middle one-third of NS5A protein. NS5A was found to bind to heterodimeric PI3K in transient expression systems and enhanced the phosphotransferase activity of p110, the catalytic subunit of PI3K. The exact mechanism by which NS5A activates PI3K is not known, but NS5A expression increased the tyrosine phosphorylation of p85 PI3K following stimulation with EGF, indicating that NS5A interaction might facilitate the activation of PI3K by upstream signaling complexes. Along these lines, NS5A and p85 PI3K appeared to form a complex with Gab1, a cellular docking protein that provides a platform for the recruitment and activation of downstream signaling molecules in the vicinity of various growth factor and cytokine receptors (He et al., 2002). Stimulation of PI3K activity by NS5A results in increased phosphorylation and activation of AKT/PKB (He et al., 2002; Street et al., 2004). NS5A expression also modulated serine phosphorylation and function of the proapoptotic protein BAD, also a direct substrate of AKT. This indicates that NS5A might modulate host cell survival and

281

HCV NS5A

contribute to HCV persistence by interacting with the PI3K-AKT cell survival pathway. Indeed, NS5A activation of the PI3K-AKT pathway correlated with the protection against apoptosis in NS5A-expressing cells or HCV replicon cells (Street et al., 2004). However, NS5A may also disrupt apoptosis through other mechanisms in these systems (see following parts in this section). In addition, expression of the HCV polyprotein in cells also activated the PI3K-AKT pathway, resulting in the modulation of two other AKT substrates, the Forkhead transcription factor and GSK-3β, indicating that HCV may affect multiple AKT-mediated pathways and biological functions (Street et al., 2005). Still, the downstream effects of the interaction between NS5A and the PI3K-AKT pathway are not completely understood and require clarification. Collectively, results from the studies reviewed here suggest a multi-faceted model of NS5A action in which NS5A is involved in the modulation of various cellular pathways. What is not so clear is which of these cellular pathways are physiologically important during HCV infection in vivo, and how the interactions between NS5A and multiple signaling pathways are coordinated and regulated during the HCV replication process.

Previous studies have shown that NS5A promotes cell proliferation resulting in cellular transformation through a PKR-dependent mechanism (Gale et al., 1999; Gimenez-Barcons et al., 2005). NS5A may also directly interact with the cell cycle control machinery. Several studies showed that NS5A repressed the expression of p21WAF1, a cell cycle regulatory gene (Ghosh et al., 2000b; Ghosh et al., 1999; Gong et al., 2004; Lan et al., 2002; Majumder et al., 2001; Qadri et al., 2002) resulting in increased cell proliferation and a transformed phenotype. The downregulation of p21 expression might involve direct NS5A interaction with SRCAP, a cellular transcription factor (Ghosh et al., 2000b), and in addition was suggested to be dependent on the tumor suppressor gene, p53 (Majumder et al., 2001). NS5A directly bound to and co-localized with p53 in the perinuclear membrane region, which may cause sequestration of p53 in this region (Lan et al., 2002; Majumder et al., 2001). NS5A inhibited the transcriptional activation activity of p53, resulting in inhibition of p21 expression, which is activated by p53 (Lan et al., 2002; Qadri et al., 2002). NS5A repression of p53 activity might involve additional factors in the p53 transcriptional activation complex. For example, NS5A was found to interact and co-localize with hTAF(II)32, a co-activator of p53. In addition, NS5A formed a heterotrimeric complex with TBP and p53 and inhibited the binding of these two proteins to their consensus DNA binding sequences (Lan et al., 2002; Qadri et al., 2002). These observations suggest that NS5A has the potential to interact with multiple cellular transcription factors and regulate the expression of cell-cycle control genes. However, in contrast to these experiments, two other studies showed that NS5A expression actually inhibited cell proliferation in various cell types, which exhibited a reduced S phase and an increase in the G2/M phase (Arima et al., 2001; Siavoshian et al., 2004). The underlying

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mechanism was suggested to be either p53-dependent induction of p21 (Arima et al., 2001), or through a p53-independent mechanism (Siavoshian et al., 2004). The underlying reason for the discrepancy in these results is not clear, but may be due to the different assay systems being utilized. Overall, the mechanistic details of how NS5A affects cell cycle control pathways are still not well understood and await further characterization in the HCV infection system.

Apoptosis is a proactive cell death process and in some cases is caused by viral infection. Prevention of host cell apoptosis may be beneficial to viruses by allowing longer periods of viral replication and persistence. It has been shown that NS5A could disrupt the apoptotic process through either PKR- or p53-dependent mechanisms (Gale et al., 1999; Lan et al., 2002). In addition, NS5A was able to inhibit apoptosis induced by treatment of human hepatoma cell lines with TNF-α. This effect correlated with a block in the activation of cellular caspases and downstream proapoptotic events (Ghosh et al., 2000a; Miyasaka et al., 2003). Interestingly, in transgenic mice expressing NS5A in the liver, TNF-induced apoptosis was prevented (Majumder et al., 2002). NS5A was found to physically associate with the TRADD signaling complex, which associates with the TNF receptor, and reduced the interaction between TRADD and FADD (Majumder et al., 2002; Park et al., 2002). So it seems that NS5A can block TNF-dependent apoptosis by associating with and disrupting the TRADD-FADD signaling complex.

In addition, it was found that NS5A expression inhibited TNF-induced activation of NK-κB, which is mediated by TRADD and TRAF2 (Majumder et al., 2002; Park et al., 2002). Consistently, NS5A directly interacts with and co-localized with TRAF2. The interaction was mapped to a.a. 148-301 of NS5A and required the TRAF-domain of TRAF2. However, NS5A did not block the recruitment of either TRAF2 or IKK-β to the TNF receptor complex, suggesting that NS5A may form a multi-subunit complex with at least TRAF2 and TRADD in the vicinity of TNF receptor (Park et al., 2003). Curiously, NS5A was also found to enhance TRAF2-mediated JNK activation by TNF-α (Park et al., 2003). It is unclear how the NS5A-TRAF2 interaction differentially modulates the NF-κB and JNK pathways. However, it is tempting to speculate that NS5A might disrupt host cell inflammatory and immune responses. What affect this might have in the context of HCV infection is not known. In addition, whether or not the NS5A-TRAF2 interaction is required for HCV RNA replication in cell culture requires further testing. Given that TRAF2 may also mediate cellular ER stress response and PKR-dependent NF-κB activation, it is possible that NS5A interaction with TRAF2 may also affect these cellular processes as well.

In another study, NS5A was able to antagonize sodium phenylbutyrate (NaPB)-induced apoptosis in hepatocellular carcinoma cells, a p53-independent process

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(Chung et al., 2003). NS5A was shown to co-localize and interact with Bax, a pro-apoptotic Bcl-2 family member, in the nucleus after NaPB treatment. Surprisingly, NS5A was found to contain a few Bcl-2 homology domains (BH3, BH1, and BH2; Fig. 1), which are domains found in Bcl-2 family members and mediate interaction between Bcl-2 proteins. BH3 and BH1 are in the N-terminal half of NS5A, while BH2 partially overlaps with the ISDR. A mutant of NS5A deleted for both BH2 and the putative NLS regions localized to the cytoplasm and disrupted its association with Bax. In addition, this mutant protein was no longer able to suppress NaPB-induced apoptosis. On the other hand, deletion of the NLS region alone resulted in a protein which still associated with Bax in the perinuclear region, but showed reduced association with Bax in the nucleus and reduced ability to block NaPB-induced apoptosis. These results suggest that NS5A may act as a Bcl-2 analogue and interact with Bcl-2 family members to block the apoptosis pathway, and this process may require the nuclear form of NS5A protein. As discussed previously, the biological function of the nuclear form of NS5A and the mechanism by which it is produced is not clear. Similarly, the role of the nuclear form of NS5A as it pertains to HCV infection requires additional experimentation.

Viral infection frequently results in activation of host cell defense mechanisms and stress responses, due to overexpression of viral proteins, stimulation of the innate immune responses pathways such as the IFN system, and disruption of normal cellular functions. In contrast to the above mentioned studies on the TNF receptor, Gong and colleagues found that NS5A expression activated the NF-κB and STAT3 transcription factors through oxidative or ER stress (Gong et al., 2001; Waris et al., 2002). NS5A seems to trigger oxidative stress by disturbing intracellular calcium pools, and the activation of NF-κB and STAT3 by NS5A is sensitive to inhibition by antioxidants and calcium chelators. Activation of the NF-κB pathway was also confirmed by microarray analysis of Huh7 cells expressing NS5A, since many NF-κB responsive genes were identified (Girard et al., 2004). The activation of the NF-κB pathway by NS5A may involve a novel mechanism involving tyrosine phosphorylation of IκB-α at two sites (Tyr42 and Tyr305) suggesting an alternative activation mechanism (Waris et al., 2003). Additionally, oxidative stress and activation of NF-κB have also been observed in HCV replicon cells, but it is unclear whether these effects are specifically caused by NS5A (Qadri et al., 2004; Waris et al., 2003). In addition, in NS5A-expressing transgenic mice, activation of the STAT3 transcription factor was also observed in the mouse liver (Sarcar et al., 2004). In this study, it was suggested that the activation of STAT3 by NS5A might involve the association of NS5A with the Jak1 kinase. It is unclear whether the NS5A-Jak1 association occurs during IFN signaling or whether this has an impact on IFN-induced antiviral responses. It is noteworthy that many of the cell culture-based studies reviewed in this chapter involve overexpression of NS5A and other HCV proteins at concentrations that are most likely higher than those in

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HCV-infected liver cells in patients. Thus, all these studies need to be considered in the proper context. Thus, future studies will more than likely be aimed at verifying these results in experimental systems that are more physiologically relevant as they become available.

In addition to the cellular signaling pathways discussed above, NS5A has also been reported to interact with a wide variety of cellular proteins. These NS5A-interacting proteins include karyopherin beta 3 (Chung et al., 2000), the adaptor protein amphiphysin II (Zech et al., 2003), the homeodomain protein PTX1 (Ghosh et al., 2003), and HSP27 (Choi et al., 2004), among many others [Table 1]. The exact physiological affects of these interactions require follow-up studies, but it seems likely that the range of cellular signaling pathways that are affected by NS5A is likely to expand.

CONCLUDING REMARKSTremendous progress has been made in our understanding of the biology of the NS5A protein. Recent biochemical and structural studies have given us great insight into the location of NS5A in various cellular compartments and the domain architecture of this protein. Various cellular binding partners have been identified and the affects of NS5A on various cellular signal transduction pathways continue to be an area of great interest. The role of phosphorylation of NS5A by host cell kinases continues to be defined. In addition, the advancement of the HCV replicon system has shed light on the physiological role of NS5A in viral replication. Despite this progress, several key questions remain. The precise role of the various forms of NS5A both in terms of subcellular localization and phosphorylation needs be systematically addressed. In addition, the role of other NS proteins in phosphorylation needs to be further refined and the clear identification of host cell kinases leading to both basal and hyperphosphorylation of NS5A requires additional work. One of the most exciting areas of research on NS5A is its interactions with host cell proteins and its ability to modulate host pathways. In most cases, the physiological role of these interactions needs to be studied in the context of viral replication. While we have learned much about the ability of NS5A to modulate the IFN response, more research is needed into its effects on other aspects of innate immunity. With the recent development of an HCV infection model, future work will most certainly be aimed at investigating the role of NS5A in other aspects of the HCV life cycle including viral entry and assembly. As new in vivo models evolve (see Chapter 12), the role of NS5A in virus replication in animal models will certainly be defined. Thus, future research should provide new clues as to the various functions of this truly remarkable multifunctional regulator.

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Chapter 10

Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase

C. T. Ranjith-Kumar and C. Cheng Kao

ABSTRACTStructural and functional studies of the hepatitis C virus (HCV) RNA-dependent RNA polymerase have contributed to our understanding of polymerase mechanism, viral RNA replication, and have generated targets for antiviral development. This review summarizes recent studies on the properties of the HCV polymerase.

INTRODUCTIONHCV, like other (+)-strand RNA viruses, uses its viral genomic RNA as a template for both translation and generation of a complementary (-)-stranded RNA intermediate. The (-)-stranded RNA is then used as the template for the synthesis of molar excess of (+)-stranded progeny RNA molecules. (For a good general review on RNA virus replication, see Buck, 1996.) A membrane-associated replicase enzyme complex consisting of virally encoded and host proteins is responsible for the replication of viral RNA. The catalytic subunit of the replicase complex is the HCV encoded nonstructural 5B protein (NS5B), which contains all the sequence motifs highly conserved among all the known RNA-dependent RNA polymerases (RdRps) (Poch et al., 1989). By extension of studies from the human immunodeficiency virus (HIV), where the reverse transcriptase is a primary target for effective antivirals, the HCV RdRp is considered an important target for drug development (Beaulieu and Tsantrizoa, 2004; Wu and Hong, 2003).

Analysis of HCV replication has been hampered by the lack of convenient animal model and efficient cell culture systems. As a result, compounds against HCV have been screened using either surrogate viruses such as bovine viral diarrhea virus (BVDV) (Bukhtiyarova et al., 2001), biochemical targets such as the NS3 protease-helicase and/or the NS5B RdRp (Sarisky, 2004), and hepatoma cell line Huh7 expressing the subgenomic replicon (Lohmann et al., 1999; Blight et al., 2000; Guo et al., 2001; Ikeda et al., 2002). Subgenomic replicons are increasingly used to screen and characterize antivirals (Horscroft et al., 2005), although inhibitors identified using such cell-based screens still need to be tested against all viral proteins encoded by the replicon system to determine the mechanism of action. Thus, the HCV NS5B remains a target of choice for both nucleoside and nonnucleoside

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inhibitors. This chapter we will focus on the structural and functional aspects of the HCV RdRp.

EXPRESSION OF NS5BExpression of recombinant NS5B in insect and bacterial cells provided valuable reagents for the biochemical characterization. NS5B expressed using the baculovirus system can perform RNA-dependent RNA synthesis (Behrens et al., 1996; Lohmann et al., 1997). However, generation of soluble full length NS5B in bacterial cells proved unsuccessful in spite of a number of attempts (Yuan et al., 1997). A hydrophobic profile of the NS5B revealed that the C-terminal 21 amino acid residues is highly hydrophobic and is predicted to insert into membrane. In fact, membrane association of the RdRp is essential for the replication of HCV subgenomic replicons in cells (Moradpour et al., 2004). Deletion of this C-terminal tail of NS5B resulted in a soluble protein that had properties similar to that of protein expressed using insect cells, indicating that the C-terminal tail contributes minimally to nucleotide polymerization (Yamashita et al., 1998). Vo et al. (2004) have evidence suggesting that the C-terminal tail in the recombinant NS5B protein will increase interaction with RNA. However, it is unclear how a hydrophobic region can affect RNA binding.

Enzymatically active NS5B proteins fused to glutathione S-transferase or a histidine tag have been generated (Yamashita et al., 1998; Oh et al., 1999; Ferrari et al., 1999). However, NS5B with a N-terminal histidine tag was expressed at a lower level and also had lower activity compared to the C-terminally tagged protein (Lohmann et al., 1997; Ferrari et al., 1999). This phenomenon may be related to the results from Lohmann et al. (1997, and 1998), who found that changes in the N-terminal residues of NS5B reduced RdRp activity in vitro (Lohmann et al., 1997 and 1998) and that residue Cys14 of NS5B contributes to RNA binding (Bressanneli et al., 1999; O'Farrell et al., 2003).

Fig. 1. A schematic of the HCV RdRp depicting the locations of the motifs and domains. The sequence alignments of the six recognizable motifs within RdRps from HCV, the double-stranded RNA phage φ6 and poliovirus 3Dpol are also shown.

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STRUCTURAL FEATURES OF HCV NS5BSimilar to other known RdRps, the HCV NS5B also contains six conserved motifs designated A-F. Comparison of the HCV NS5B sequence with the poliovirus RdRp and the φ6 RNA polymerase, two other model RdRps, is shown in Fig. 1. The three-dimensional crystal structure of HCV NS5B has been determined independently by several groups (Lesburg et al., 1999; Bressanelli et al., 1999; Ago et al., 1999). Using the right-hand analogy for polymerases (Joyce and Steitz, 1995), the HCV RdRp has discernable fingers, palm and thumb subdomains. An unusual feature of this polymerase is that, due to the extensive interactions between the finger and thumb subdomains, the HCV RdRp has an encircled active site (Fig. 2) (Lesburg et al., 1999; Bressanelli et al., 1999; Ago et al., 1999). These contacts restrict the flexibility of the subdomains and possibly constrain flexibility between the subdomains. The HIV reverse transcriptase and other DdRps are known to undergo transition from an open or to closed conformation upon template binding and polymerization (Doublie et al., 1999; Huang et al., 1998). Structural analysis of HCV NS5B (J4 strain) revealed that de novo initiation is the probable mode of RNA synthesis, and limited structural changes take place upon nucleotide binding

Fig. 2. The structure of the HCV RdRp. The figure is from Lesburg et al. (1999), reprinted with the permission of the publisher and modified to denote the positions of the thumb, finger and palm domains, and the location of the template channel. The colored structures denote conserved motifs within the RdRp: red, motif A; green, motif B; yellow motif C; light violet,motif D; purple: motif E; dark grey, motif F. The β-loop is in light brown.A colour version of this figure is printed in the colour plate section at the back of this book.


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(O'Farrell et al., 2003). Structural studies with RdRp from the HCV genotype 2a indicate the presence of two conformations of the protein even in the absence of template RNA, where the key difference between the two forms is the relative orientation of the thumb domain in relation to fingers and palm domains (Biswal et al., 2005). Since both conformations lacked RNA, whether the template RNA will induce the same structural change(s) remains to be determined.

Another unusual feature of NS5B is a β-hairpin loop that protrudes into the active site located at the base of the palm subdomain (Fig. 2). This 12 amino acid loop was suggested to interfere with binding to double stranded RNA due to steric hindrance (Hong et al., 2001). The poliovirus RdRp lacks a similar β-loop, a feature that may be related to the poliovirus RdRp normally directing genome replication with a protein-nucleotide primer (Paul et al., 1998). It was suggested that the β-loop could be involved in positioning the 3' terminus of the viral RNA for correct initiation (Hong et al., 2001). Since the wild-type HCV RdRp is fully capable of primer-dependent RNA synthesis, additional factors are needed to prevent primer extension. Indeed, GTP can, along with structures within the RdRp, help prevent primer-extension (Ranjith-Kumar et al., 2003).

The C-terminal tail of NS5B also lines the RNA binding cleft in the active site. This region, which immediately precedes the C-terminal membrane anchorage domain, forms a hydrophobic pocket and interacts extensively with several important structural elements including the β-loop (Adachi et al., 2002; Leveque et al., 2003).

Fig. 3. The catalytic NTP binding pocket in the HCV RdRp. The structure is from Bressanelli et al. (2002) and is reprinted with the permission of the publisher. Left panel: a detailed view of the NTP binding site in complex with rUTP. Amino acids involved in binding NTP are labeled. Divalent metals are depicted as grey spheres labeled with A and B. Right panel: a detailed view of the active site of the HCV RdRp with the potential entry portal for incoming NTPs.A colour version of this figure is printed in the colour plate section at the back of this book.

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Deletions of up to 55 residues of the C-terminal tail resulted in increased RdRp activity, suggesting a direct role for the C-terminal tail in RNA synthesis activity and providing evidence for regulation at the active site (Lohmann et al., 1997; Tomei et al., 2000; Ranjith-Kumar et al., 2002).

CATALYTIC POCKETThe active sites of HCV NS5B and HIV-1 reverse transcriptase are very similar and can be superimposed without significant steric clashes (Lesburg et al., 1999). The residues involved in nucleotidyl transfer are found in palm motifs A and C. Motif A harbors the metal binding residue D220 which is a part of conserved D-X4-D motif, while motif C has the conserved metal binding and nucleotidyl transfer residues D318 and D319 (Fig. 3). D225 within motif A forms a H-bond with the ribose 2'-hydroxyl group of the NTP and is thought to discriminate against the use of dNTPs. Crystal soaking experiments with HCV NS5B with NTPs revealed several residues in the catalytic pocket that contact the triphosphates of NTP (Fig. 3). These include R158, S367, R386, T390 and R394. Unfortunately, it was not possible to identify the base-interacting residues. The role of these residues in RdRp activity is discussed in more detail later in this chapter.

Soaked NS5B crystals with GTP also revealed a low affinity GTP binding site on the surface of the protein (Fig. 4; Bressanelli et al., 2002). This second site, which is apparently specific for GTP, lies between the fingers and thumb subdomains, approximately 30 Å away from the catalytic pocket. By virtue of its position and the

Fig. 4. A low affinity rGTP binding site in the HCV RdRp. The structure is from Bressanelli et al. (2002) and reprinted with the permission of the publisher. Left panel: a view of the back of the HCV RdRp emphasizing the low affinity GTP binding pocket. The arrows denote β-strands, the cylinders denote helices and lines denote connecting loops. The low affinity GTP binding site resides in the blue colored region containing the thumb subdomain. Right panel: a more detailed view of the low affinity rGTP binding pocket.A colour version of this figure is printed in the colour plate section at the back of this book.


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requirement of higher GTP concentrations to saturate the pocket, it was suggested that it might play a regulatory role in RNA synthesis (Bressanelli et al., 2002). To date, any allosteric properties of this low affinity site in response to GTP has not been determined.

ACTIVITIES OF NS5BRecombinant NS5B was sufficient to synthesize full length HCV RNA in vitro (Hwang et al., 1997; Yamashita et al., 1998; Behrens et al., 1996; Lohmann et al., 1997; Ferrari et al., 1999). This observation indicates that NS5B can also unwind stable secondary and tertiary RNA structures. As covered in Chapter 2 of this book, the 5' and 3' untranslated regions (UTRs) of the HCV genome contain highly ordered and complex RNA structures , which are highly conserved and contain cis-acting elements for viral RNA replication. Recently it was shown that a pseudoknot structure is formed between the 3' end of the HCV genome and a novel RNA element in the NS5B coding sequence (Friebe et al., 2005). The 3' terminal 150 nt of the HCV RNA contain signals that are essential for RdRp binding and replication of viral RNA (Yi and Lemon, 2003a and b; Reigadas et al., 2001; Cheng et al., 1999). In vitro HCV NS5B was found to replicate the 3' terminal region of the (-)-strand RNA more efficiently than the 3' terminal region of (+)-strand RNA (Reigadas et al., 2001). Analysis of the promoter elements in 3' terminus of (-)-strand revealed that complementary strand of second stem-loop of the internal ribosome entry sequence (IRES) binds NS5B and acts as a positive element for RNA synthesis (Kashiwagi et al., 2002). However, the complementary strand of the first stem-loop of the IRES worked as a negative regulator of RNA synthesis (Kashiwagi et al., 2002).

Though some genome specific recognition was observed, recombinant NS5B largely lacked specificity for binding to HCV RNA (Lohmann et al., 1997). Several groups used different RNAs to analyze RdRp activity. In general, the HCV NS5B preferred a template with a stable 5' secondary structure(s) and a single stranded sequence that contained at least one 3' cytidylate (Kao et al., 2000). This observation was further supported when high-affinity RNA ligands to HCV NS5B were isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure (Vo et al., 2003). The high affinity RNA was found to have three stem-loop structures.

Our lab uses short RNAs to study de novo initiation of RNA synthesis by the HCV NS5B because products from these templates can be identified with single-nucleotide resolution. The prototype RNA, LE19, was derived from BVDV. LE19 is predicted by mfold to form a stem-loop with five intramolecular base pairs and with single-stranded sequences of three nucleotides at both the 5' and 3' ends (Fig. 5). The 3' sequence contains a cytidylate that can be used as an initiation nucleotide. Two LE19 molecules also form a heterodimer which can be extended to form a 32-nt primer

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extension product. In addition, NS5B can add nontemplated nucleotides to the RNA, a process known as terminal nucleotidyl transferase (TNTase). Lastly, HCV RdRp can generate recombinant RNA product from two or more non-covalently linked templates, a process known as template switch. All these activities can be studied using LE19 in a single reaction (Fig. 5). The different activities of NS5B and their potential importance in viral replication are discussed in detail below.

DE NOVO SYNTHESIS AND PRIMER EXTENSIONInitiation of RNA synthesis in infected cells likely starts de novo, by use of a one-nucleotide primer (Bressanelli et al., 2002; Ferrari et al., 1999). This process is well studied in DNA-dependent RNA polymerase, which uses two NTP binding sites in

Fig. 5. An RNA template that can be used to examine several activities of the HCV RdRp in one reaction. A) Schematics of the various activities of the HCV RdRp using LE19. LE19 exists in an equilibrium between monomeric and dimeric forms. De novo initiation occurs at the 3'- terminal cytidylate of the monomer to generate a 19-nt product. Two LE19 molecules could base-pair through the nucleotides at their 3'-termini to generate templates for primer extension. In addition, de novo initiation from one template could result in a ternary complex that does not terminate, but instead uses a second template to form a template switch product. Lastly, the 3' terminus of LE19 could act as the acceptor for nontemplated nucleotide addition. B) A demonstration of the effects of GTP and Mg2+ on the activities of the HCV RdRp. The autoradiogram shows the products synthesized by the HCV RdRp Δ21 or Δ21 with mutations in the divalent metal-binding residues (D318 and D319). The presence or absence of GTP are noted with "+" and "-", respectively. The length of the RNAs (in nucleotides), and the mechanism used to generate a product are noted to the right and left of the autoradiogram, respectively.


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the catalytic pocket. The first site is called the I site and specifically recognizes the initiating NTP (NTPi). The second site, the I+1 site, is less specific and recognizes the NTP complementary to the second template nucleotide. The flaviviral RdRps can also accommodate two NTPs, with the I site preferentially binding to GTP (Ferrari et al., 1999; Kao et al., 1999; Lohmann et al., 1999; O'Farrell et al., 2003; Oh et al., 1999; Luo et al., 2000; Ranjith-Kumar et al., 2002; Sun et al., 2000; Zhong et al., 2000; Zhong et al., 2000). The catalytic aspartates coordinate divalent metals that are in position to help form a phosphodiester bond between the NTPi and the second NTP (Ferrari et al., 1999; O'Farrell et al., 2003).

While GTP is generally accepted as the NTPi for RNA synthesis by the HCV RdRp in vitro, the 3' terminal residue of many HCV isolates is a uridylate, suggesting that ATP may be used to initiate (-)-strand RNA synthesis. Recently, a subgenomic replicon that was passaged in vitro was demonstrated to switch from using GTP to ATP as the NTPi for both (+)- and (-)-strand RNA replication (Cai et al., 2004). The exact identity of the NTPi is specific for a purine triphosphate, but can be somewhat flexible, as it is for DNA-dependent RNA polymerases (Kuzmine et al., 2003 and references within).

Polymerases require divalent metal ions for activity. RNA synthesis by NS5B is increased by 4-20 fold when Mn2+ is present in the reaction in comparison to a reaction with only Mg2+ (Zhong et al., 2000; Ferrari et al., 1999; Luo et al., 2000; Ranjith-Kumar et al., 2002). However, other divalent metal ions such as Co2+, Cu2+, Ni2+ and Zn2+ did not support RdRp activity (Luo et al., 2000; Ranjith-Kumar et al., 2002). Recently it was shown that iron binds specifically to the Mg2+ binding site of NS5B and can inhibit RNA synthesis (Fillebeen et al., 2005). Mn2+ appears to more specifically contribute to de novo initiation by lowering the KM for GTP by about 30-fold (Ranjith-Kumar et al., 2002). While the concentration of Mn2+ used in vitro is far higher than concentrations present in the cell, physiologically relevant Mn2+ levels can increase de novo initiation with GTP (Ranjith-Kumar et al., 2002). Analysis of proteins with C-terminal deletions revealed that the C-terminus of the HCV RdRp plays a role in Mn2+ induced de novo initiation and can contribute to the suppression of primer extension (Ranjith-Kumar et al., 2002). Spectroscopy examining the intrinsic tryptophan and tyrosine fluorescence of the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of divalent metals (Ranjith-Kumar et al., 2002; Bougie et al., 2003).

Even though de novo initiation seems to be the preferred mechanism of initiation, it has always been puzzling that primer-extension activity was far more robust than de novo initiation in vitro. This begs the question of whether the two mechanisms will affect each other. Deletion of the β-loop led to an increase in primer extension activity (Hong et al., 2001). However, this deletion was only part of the requirement

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for the suppression of primer extension since additional characterizations revealed that the NTPi-binding site, higher concentrations of the initiation GTP, and the C-terminal tail that lines the catalytic pocket all participate to suppress primer extension (Ranjith-Kumar et al., 2003). Thus, the features and requirements for de novo initiation by the HCV RdRp significantly prevent primer extension.

MUTATIONAL ANALYSISSite directed mutagenesis has been employed to study the role of specific residues in RdRp activities (Lohmann et al., 1997, Qin et al., 2001; Labonte et al., 2002; Ranjith-Kumar et al., 2002, 2003 and 2004). Mutation of the conserved catalytic residues D318 and D220 resulted in inactive proteins. Lohmann et al. (1997) investigated the effects of mutations of some conserved residues in motifs A, B, C and D. Most of the mutations on the conserved residues in motifs A and B led to inactive protein and inactive subgenomic replicons in cell culture (Cheney et al., 2002). However, mutations of the conserved residues G317, and D319 in motif C were tolerated somewhat. Interestingly, substitution of R345 with lysine in motif D enhanced the enzymatic activity (Lohmann et al., 1997). A similar increase in activity was observed when K151 was mutated to glutamate (Labonte et al., 2002). Qin et al., (2001) generated a series of clustered and point mutations and studied their effects on RdRp activity and template binding. The residues that affected RdRp activity included E18, Y191, C274, Y276 and H502. Y276 was also found to be important for interaction with the template/primer.

The structures of the HCV RdRp and nucleotides identified a number of interacting residues at D225, R48, R158, R386, R394, and S367 that interact with the initiation GTP (Bressanelli et al., 2002; Fig. 3). In this structure, it is not clear whether the GTP is binding to the I site or the I+1 site. NTPi binding to the I site is base-specific while binding of the second NTP to the I+1 site should be directed by the template (Ranjith-Kumar et al., 2002). Because the RdRp-GTP structure was determined without a template, it is likely that the residues identified recognized the NTPi. Alanine substitutions of these residues were analyzed for effects on de novo initiation, primer extension, TNTase, and template switch (Ranjith-Kumar et al., 2004). Although all mutations retained the capability for primer extension, alanine substitutions at R48, R158, R386, R394, and D225 decreased de novo initiation, and two or more mutations in combination abolished de novo initiation (Ranjith-Kumar et al., 2004). It is likely that these mutations affected the stability of the initiation complex, since many of the defects were rescued when the reactions were supplemented with Mn2+. We note that several of the mutant enzymes were selectively affected for de novo initiation and/or terminal nucleotide addition, indicating that the residues in the active site can contribute differentially to the known activities of the HCV RdRp. Furthermore, while the prototype enzyme had a KM for GTP of 3.5 μM, all mutations except one negatively affected the KM


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for GTP by 3 to 7 fold, demonstrating that the affected residues are functionally required to interact with the initiation nucleotide. Lastly, mutations in D225 are dramatically affected in template switch, suggesting that this residue of the NTPi pocket also participates in the elongation complex.

TERMINAL NUCLEOTIDYL TRANSFERASE (TNTase) ACTIVITYTNTase activity could be a significant concern in biochemical screens where the products of the HCV RdRp are not visualized by denaturing gel electrophoresis. This activity was observed by some (Behrens et al., 1996; Ishii et al., 1999; Ranjith-Kumar et al., 2001), but not by others, leading some to suggest that it should be attributed to cellular contaminants rather than the HCV RdRp (Lohmann et al., 1997, Oh et al., 1999; Johnson et al., 2000; Zhong et al., 2000). Ranjith-Kumar et al. (2001; 2003) demonstrated that the HCV NS5B does have TNTase activity. First, mutation of the conserved GDD motif inactivated both polymerase and TNTase activities. Second, a HCV NS5B specific inhibitor inhibited both activities. Third, proteins purified from eukaryotic and prokaryotic expression systems both showed TNTase activity. Fourth, several mutations in the residues in the HCV RdRp that affected de novo initiation also affected TNTase activity, implicating the NTPi pocket in TNTase activity. Consistent with this last claim, Mn2+, which increases de novo initiation, also increases TNTase activity (Ranjith-Kumar, 2004). Perhaps one reason for the discrepant observations of TNTase activity from different laboratories is that the amount of TNTase depends on the template sequence and the NTPs used (Ranjith-Kumar et al., 2001).

TEMPLATE SWITCHRNA recombination contributes to genetic diversity and pathogenesis of RNA viruses (Nagy and Simon, 1997; Jarvis and Kirkegaard, 1991). HCV NS5B can generate RNA products that are larger than the size of the template RNA by a process wherein the RdRp ternary complex does not terminate RNA synthesis from a template, but will bind to a second template and continue RNA synthesis. Template switch by the related BVDV RdRp and replicase complexes from plant viruses have been characterized and require the template initiation cytidylate as well as the NTPi (Kim et al., 2001).

Several of the mutations in the NTPi pocket of the HCV RdRp affected template switch (Ranjith-Kumar et al., 2004). A defect in the template switch is to be expected with many of the NTPi mutants since fewer ternary complexes are available. However, mutant D225A, which was capable of robust de novo initiation from the first template, was debilitated for template switch in comparison to the wild-type RdRp (Ranjith-Kumar et al., 2004). We hypothesize that D225, which recognizes the ribose 2' hydroxyl of the NTPi, also plays an additional role either in recognition of the second template or in the release of the nascent RNA.

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INTERACTION WITH OTHER HCV NONSTRUCTURAL PROTEINSHCV replication uses a replicase complex that is associated with the endoplasmic reticulum (ER) (Hwang et al., 1997). The complex is thought to contain both host and virally encoded proteins, although detailed information concerning the composition of the replicase remains to be determined. A growing number of cellular proteins that could affect HCV replication activity have been identified. For example, it was recently shown that NS5B is phosphorylated by a protein kinase C-related kinase, PRK2, and this phosphorylation is involved in regulating HCV replication (Kim et al., 2004). (For general reviews of cellular factors involved in (+)-strand RNA virus infection, please see Lai et al., 1998, and Kushner et al., 2003).

HCV nonstructural proteins are co-localized with NS5B within the ER membrane and likely modulate NS5B activities (Behrens et al., 1996; Brass et al., 2002; Egger et al., 2002; Hwang et al., 1997; Wolk et al., 2000). One case is that the HCV NS5B can form oligomers in vitro and may catalyze RNA synthesis in a cooperative manner (Wang et al., 2002; Qin et al., 2002). While the C-terminal tail is not involved in this process, it was shown that two amino acids, E18 and H502, are very critical for oligomerization (Qin et al., 2002). While NS5B oligomerization can be demonstrated by several independent assays, its role in the infected cell remains to be determined.

Other interactions between HCV NS5B and other HCV nonstructural proteins have already been reported with low-resolution assays, such as protein pull-down experiments, co-immunolocalization, and yeast two-hybrid experiments. These results indicate that NS5B likely binds to NS3, and that NS3 will interact with NS4A and possibly NS4B and NS5B (Piccininni et al., 2002). NS5A may interact with NS2, NS3, NS4A, NS4B, NS5B and with itself (Dimitrova et al., 2003, Shirota et al., 2002). The NS5A protein is important for HCV replication and mutation that can increase the efficiency of subgenomic replicon replication can be mapped to it (Blight et al., 2000; Krieger et al., 2001). By using GST pull down and coimmunoprecipitation assays, NS5A was shown to directly interact with NS5B and modulate its activity (Shirotta et al., 2002).

NS3 is a multifunctional protein possessing protease, helicase, and NTPase activities (Borowski et al., 2002). Recently, the NS5B protein was shown to bind to NS3 through the protease domain and increase the helicase activity of NS3 by approximately five fold (Zhang et al., 2005). These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, full-length NS3 reduced RNA synthesis by the NS5B RdRp in vitro, possibly due to the NTPase activity of NS3 degrading NTPs nonspecifically. Whether this effect is relevant in vivo is unclear, and additional regulations of NS5B activity in the context of the HCV replicase remain to be characterized.


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A major hurdle in understanding the HCV replication complex is that the replicase is not only present in relatively low abundance, but enriched replicase can only perform elongative RNA synthesis from the template RNA that copurified with the enzymatic activity (Hardy et al., 2003; Lai et al., 2003, Ali et al., 2002). These features have made the replicase a poor reagent to understand the requirements for HCV replication in vitro. Nonetheless, the recent demonstration of the ability to produce HCV elongation-competent replicase is an important first step in its characterization and could provide a useful reagent to characterize drugs identified in cell-based screens.

CONCLUSION AND FUTURE TRENDSAlthough the initial effort to characterize the HCV RdRp was in response to a need to develop biochemical targets against HCV, recent research by the virology and structural biology communities has developed a strong basic understanding of the mechanism of action of a viral RNA-dependent RNA synthesis. This information should also prove useful in understanding the mechanism of action of antivirals targeted against HCV replication. Despite the progress made, much remains to be done. One bottom line is that we still do not have an effective drug targeting the HCV RdRp. In terms of future biochemical analyses, we need to: 1) gain a deeper understanding of the mechanism of polymerization and the conformational changes as the polymerase goes from initiation to elongative synthesis to the termination of RNA synthesis. Toward this effort, a true ternary complex at high resolution will be critical; and 2) define the effects of other viral and cellular subunits that participate in RNA-dependent RNA replication. Since HCV replication is a process intimately associated with the cellular membranes, understanding HCV replication will benefit from an infusion of expertise from membrane biologists and biochemists, and scientists trained to study the mechanism of complex enzymes, including those who have analyzed nucleic acid synthesis from other template-dependent polymerases. It is such multidisciplinary approach that could ultimately yield significant benefits to society against a disease with enormous worldwide impact.

ACKNOWLEDGEMENTSWe thank our colleagues at Texas A and M University for helpful discussions and Y. Kim for editing this manuscript. The Kao lab is supported by the National Science Foundation MCB grant 0332259.

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Chapter 11

HCV Replicon Systems

Keril J. Blight and Elizabeth A. Norgard

ABSTRACTWith the remarkable ability of hepatitis C virus (HCV) to establish persistent infections that can lead to progressive liver pathology and the poor response of prevalent HCV genotypes to the current treatment, HCV represents a significant global health problem. Studies of HCV replication in cell culture were virtually impossible until the development of subgenomic replicons that replicate autonomously in the human hepatoma cell line Huh-7. Many improvements to the replicon system have been made allowing the establishment of transient replication assays for HCV genotypes 1a, 1b, and 2a. Specifically, the identification of adaptive mutations that drastically enhance HCV genotype 1 replication and the isolation of highly permissive Huh-7 sublines led to the development of replication-competent full-length genomes in addition to a collection of robustly replicating subgenomes derived from genotype 1 sequences. More recently, the cell tropism of HCV subgenomic replicons has been expanded to non-hepatoma cell lines and mouse hepatocytes. The HCV replicon system has opened new avenues for detailed molecular studies of RNA replication and HCV-host interactions as well as the development of active inhibitors of HCV replication. Finally, the identification of genotype 2a-derived replicons that efficiently replicate in cell culture without adaptive mutations has facilitated the development of systems supporting the complete virus life cycle.

INTRODUCTIONPersistent infection with HCV has emerged as one of the primary causes of chronic liver disease, with an estimated 170 million carriers throughout the world (WHO, 2000). Viral persistence develops in ~80% of infected individuals and although the acute phase of infection is frequently asymptomatic or associated with mild and non-specific symptoms, these patients are at risk for developing chronic liver disease (Alter and Seeff, 2000). Approximately 20% of chronic carriers will develop cirrhosis, and some of these cases will progress to hepatocellular carcinoma. Consequently, HCV-induced chronic liver disease is now recognized as the leading indication for orthotopic liver transplantation in the United States (Fishman et al., 1996).

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Fig. 1. Structure of the HCV genome and Con1-derived bicistronic replicon, location of adaptive mutations in the HCV polyprotein of subgenomic replicons, and the positions of these mutated residues in the crystal structures of the NS3 protease, the NS3 helicase, and the NS5B RdRp. (A) (Top) Schematic of the complete HCV genome. The 5' and 3' NTRs flank the ORF (open box) with the structural proteins located in the N-terminal portion of the polyprotein and the remainder encoding the non-structural proteins. (Bottom) Structure of the selectable Con1 bicistronic replicon composed of the 5' NTR, the first 12 amino acids of the capsid-coding region (open box) fused to the Neo gene

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Hepatitis C viruses have a high level of genetic heterogeneity and thus have been grouped by their degree of sequence identity into six separate genotypes and further divided into numerous subtypes (Simmonds et al., 1993; Robertson et al., 1998). Geographic distribution and responses to current therapy differ between genotypes. Genotypes 1a and 1b are the most prevalent in the United States and Western Europe, followed by infections with genotype 2 and 3 strains. The only licensed therapy for chronic hepatitis C infection is polyethylene glycol (PEG)-conjugated interferon (IFN)-α given in combination with the guanosine analog ribavirin; while ~90% of patients persistently infected with HCV genotypes 2 and 3 clear the virus, only 50% of patients infected with HCV genotypes 1, 4, 5, and 6 mount a sustained response (Poynard et al., 2003). Clearly, there is a need for the development of more effective therapeutic strategies to improve the clinical treatment of HCV-associated hepatitis.

HCV has been classified as the sole member of the genus Hepacivirus within the Flaviviridae family, which also includes the classical flaviviruses, such as West Nile and yellow fever viruses, and the animal pestiviruses, such as bovine viral diarrhea virus (BVDV). Like these related viruses, HCV is enveloped with a positive-sense, single-stranded RNA genome. The HCV genome is ~9.6 kb in length and consists of a 5' non-translated region (NTR) and a long open reading frame (ORF) encoding all the virus-specific proteins followed by a 3' NTR, comprised of a short variable sequence, a poly(U)/polypyrimidine [poly(U/UC)] tract, and a highly conserved terminal sequence (Fig. 1A). Translation of the genomic RNA is mediated by an internal ribosome entry site (IRES) located within the 5' NTR (reviewed in Rijnbrand and Lemon, 2000). The resulting polyprotein precursor of about 3000 amino acids is co- and post-translationally cleaved into at least 10

(Neo; black box), the EMCV IRES (EMCV; solid line), the NS3-5B coding region (open box), and the 3' NTR structure. (B) Schematic representation of the NS3 to NS5B coding region, sufficient for autonomous replication of subgenomic replicons in Huh-7 cells. The protease (P) and helicase (H) domains of NS3 are indicated along with the locations of the ISDR (shaded box) and the adaptive 47 amino acid deletion. The highly adaptive mutations in NS4B, NS5A, and NS5B and the amino acid substitutions in NS3 that act synergistically with these mutations to enhance subgenomic replication are shown to the right of the HCV NS3-5B polyprotein. The highly adaptive S2204I substitution in NS5A is highlighted in bold and those residues in NS3 (E1202G and T1280I) and in NS4B (K1846T) or NS5A (S2197P) that synergistically enhance replication are shown in italics. (C) Space-filling view of the NS3/4A serine protease (Protein Database 1A1R; Kim et al., 1996). The adaptive mutations depicted in panel B, the position of the active-site residues and the location the NS4A cofactor peptide are indicated. (D) The 3D structure of the NS3 helicase domain complexed with a single-stranded DNA oligonucleotide (Protein Database 1A1V; Kim et al., 1998). The oligonucleotide, the conserved DECH motif implicated in coupling NTP hydrolysis to nucleic acid unwinding, and the adaptive mutations are highlighted. Residue G1304 is partially buried in the molecule and therefore is not visible in the space-filling model shown. (E) Space-filling model of the NS5B RdRp ectodomain (Protein Database 1C2P; Lesburg et al., 1999). The putative RNA binding groove is labeled and the adaptive mutation is highlighted. The color version of this figure can be found at http://blightlab.wustl.edu/chapter11/.

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different products by a combination of host cell signal peptidases and two viral proteases. At the N-terminus are the structural proteins C (capsid), E1, and E2 (envelope glycoproteins) followed by the short hydrophobic peptide, p7. The C-terminal two-thirds of the polyprotein comprise the non-structural proteins (NS) 2, 3, 4A, 4B, 5A, and 5B (Fig. 1A). In addition, the frameshift (F) or alternative reading frame protein (ARFP), encoded in an overlapping reading frame within the N-terminus of the HCV polyprotein, is synthesized by ribosomal frameshift (Walewski et al., 2001; Xu et al., 2001; Varaklioti et al., 2002).

As discussed later, the non-structural proteins NS3-5B are sufficient for subgenomic replicon replication in cell culture (Lohmann et al., 1999). These proteins are presumed to function as structural and enzymatic components of the HCV replication complex or replicase, and the enzymatic properties of the non-structural proteins are fairly well defined (Table 1 and reviewed in Reed and Rice, 1999; Blight et al., 2002a). The N-terminus of NS3 is a serine protease that forms a stable complex with its cofactor NS4A to mediate cleavage of the HCV polyprotein at the NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions. The C-terminal two-thirds of NS3 harbors an RNA nucleoside triphosphatase (NTPase)/helicase activity capable of unwinding nucleic acid duplexes. How the NS3 helicase/NTPase contributes to the RNA replication process is currently unknown, although it is thought to unwind regions of extensive secondary structure in the template or double-stranded RNAs resulting from synthesis of the complementary negative-sense RNA intermediate (see below). NS5B, the C-terminal cleavage product of the polyprotein, is the RNA-dependent RNA polymerase (RdRp). The roles of NS4B and NS5A in RNA replication, however, are less clear. NS4B is an integral membrane protein that induces a distinct membrane alteration, designated the membranous web (Egger et al., 2002). NS5A is phosphorylated predominantly on serine residues by one or more unidentified cellular kinases producing two NS5A phosphoprotein variants; basal- (p56) and hyper- (p58) phosphorylated forms of NS5A (Kaneko et al., 1994; Tanji et al., 1995; Reed et al., 1997). Surprisingly, NS2 was not required for subgenomic replication in cell culture (Lohmann et al., 1999) and thus the role of NS2 in the viral life cycle is not completely clear, although it has been shown to

Table 1. Summary of the enzymatic functions of the HCV non-structural proteins.Function

NS2 autoprotease with the N-terminus of NS3 (NS2-3)NS3 serine protease (N-terminus); NTPase/helicase (C-terminus)NS4A cofactor for NS3 serine proteaseNS4B unknownNS5A unknownNS5B RNA-dependent RNA polymerase (RdRp)

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form a distinct autoprotease with the serine protease domain of NS3 (NS2-3; Table 1) responsible for cleavage at its own C-terminus (Grakoui et al., 1993; Hijikata et al., 1993).

The mechanisms of viral attachment and cellular entry and the precise intracellular steps in HCV RNA replication, virus assembly, and virion release are largely unknown due to the previous lack of suitable cell culture systems for HCV. Some details have begun to emerge since the development of subgenomic replicons that recapitulate the intracellular steps of RNA replication (Fig. 4). Briefly, input positive-sense HCV RNA is translated and the resultant polyprotein is processed into the individual HCV proteins. The non-structural proteins assemble in association with intracellular membranes into the replication complex that transcribes the input RNA molecule to generate a complementary negative-sense RNA intermediate that presumably remains base-paired with its template. This double-stranded replicative form is transcribed asymmetrically, leading to the preferential accumulation of positive-sense RNAs that are then available for further translation or synthesis of the negative-sense RNA intermediate.

Since the molecular cloning of the HCV genome 16 years ago, there has been a great deal of progress in defining HCV genome structure and protein function. However, the lack of a reliable and robust cell culture system has presented a major obstacle to studies on the viral life cycle and for developing effective antiviral drugs. These hurdles have been overcome by the development of subgenomic and genomic replicons for HCV. In this chapter, we will describe the development of the HCV replicon system along with the most recent advances and applications of this system.

DEVELOPMENT OF HCV REPLICONSNumerous attempts have been made to propagate HCV in cell culture by infection with virus-containing inoculum. Replication has been detected in hepatoma, B- and T-cell lines, primary cultures of human or chimpanzee hepatocytes, and peripheral blood mononuclear cells; however, replication levels were frequently transient and always so low that HCV RNA synthesis could only be monitored by highly sensitive reverse transcription (RT)-PCR assays and thus were not amenable to detailed studies of HCV replication (reviewed in Bartenschlager and Lohmann, 2001). For many positive-sense RNA viruses, including the closely related flavi- and pestiviruses, productive replication can be efficiently launched by transfecting permissive cells with genomic RNAs transcribed in vitro from cloned virus genomes. With this approach, the viral entry and uncoating steps are bypassed; however, transfection of HCV RNAs transcribed from cDNA clones with proven infectivity never reproducibly established HCV replication in the many cell lines tested (reviewed in Bartenschlager and Lohmann, 2001). This was most likely

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due to the generally low levels of replicated RNA and was further complicated by the difficulty of distinguishing input RNA from plus strands produced by RNA replication (Fig. 4).

Although HCV is notoriously difficult to grow in cell culture, HCV subgenomic replicons that efficiently replicate in a human hepatoma cell line, Huh-7, have been developed (Lohmann et al., 1999). The development of this system was inspired by the observation that the structural proteins were not required for replication of several positive-sense RNA viruses including flavi- and pestiviruses, and by the successful design of self-replicating replicons for the flavivirus Kunjin (Khromykh and Westaway, 1997) and the pestivirus BVDV (Behrens et al., 1998). The first generation functional HCV replicons were derived from the consensus Con1 cDNA that was isolated from the liver of a patient chronically infected with a genotype 1b strain and comprised: (i) the HCV 5' NTR and the first 12 codons of the capsid protein fused in-frame with the selectable marker gene, neomycin phosphotransferase (Neo), which upon expression confers resistance to the cytotoxic drug G418; (ii) the IRES element from encephalomyocarditis virus (EMCV), which directs translation of the HCV non-structural proteins; and (iii) the HCV 3' NTR. Fig. 1A depicts the structure of the bicistronic replicon encoding the HCV polyprotein NS3-5B. Transfection of Huh-7 cells with transcripts synthesized in vitro and selection with G418 resulted in a low number of surviving cell colonies. Independent G418-resistant cell colonies harbored replicons that replicated RNA to high levels (1,000-5,000 copies of positive-sense HCV RNA per cell), while the negative-sense replicative intermediate was 5-10-fold lower, consistent with asymmetric RNA synthesis. Autonomous HCV replication was verified by the efficient labeling of HCV RNA with [3H] uridine in the presence of actinomycin D, an inhibitor of DNA- but not RNA- dependent RNA polymerases (Lohmann et al., 1999). HCV non-structural proteins in G418-selected cell clones localized exclusively to the cytoplasm in close association with membranes of the endoplasmic reticulum (ER) (Pietschmann et al., 2001; Mottola et al., 2002), a predicted site of HCV RNA replication (discussed below).

IDENTIFICATION OF ADAPTIVE MUTATIONSDespite the high levels of subgenomic RNA replication within a selected cell clone, G418 resistance arose in a very low frequency of transfected cells (~1 colony per 106 transfected cells; Lohmann et al., 1999; Blight et al., 2000). This was due to two restrictions: first, replicon RNAs had to acquire adaptive mutations to efficiently replicate in the Huh-7 cell line; and second, only a low number of cells in the culture support efficient HCV replication (discussed in detail below). Sequence analysis of Con1-derived HCV RNAs replicating in cell clones after G418 selection identified in most cases at least one mutation in the non-structural coding region, but not in the highly conserved 5' or 3' NTRs (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). The

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impact of individual mutations on HCV RNA replication was tested by engineering mutations into the parental replicon and determining the number of G418-resistant colonies after transfection of a defined amount of in vitro transcribed RNA, or by transient replication assays. As discussed below, most mutations were found to enhance RNA replication in Huh-7 cells; however, the degree of adaptation was highly dependent on the particular substitution.

Highly adaptive mutations lie within the NS4B, NS5A, and NS5B coding regions, with the majority clustering in NS5A, just upstream of the sequence termed the IFN sensitivity-determining region (ISDR; Fig. 1B), a region that has been implicated in the effectiveness of IFN treatment (Enomoto et al., 1995; Enomoto et al., 1996). Highly adaptive amino acid substitutions have been identified at nine positions in Con1 NS5A (Fig. 1B; Blight et al., 2000; Krieger et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). The most efficient adapted replicon contains a single serine to isoleucine substitution at position 2204 in NS5A (S2204I; Fig. 1B) and establishes replication in ~10% of transfected Huh-7 cells (Blight et al., 2000). The >10,000-fold improvement in colony-forming efficiency compared to the parental Con1 replicon was sufficient for the detection of HCV RNA and proteins shortly after RNA transfection. By 96 hours, HCV RNA levels were almost 500-fold higher than a replicon carrying a lethal mutation in the NS5B RdRp (Blight et al., 2000). In addition to amino acid substitutions, an in-frame deletion of 47 amino acids encompassing the putative ISDR (∆2207-2254; Fig. 1B) (Blight et al., 2000) and a deletion of the serine residue at position 2201 (∆S2201; Fig. 1B) (Guo et al., 2001) also enhance replicon replication in Huh-7 cells.

Based on the predicted topology of the integral membrane protein NS4B, amino acid substitutions reside in distinct cytoplasmic domains of this protein (Guo et al., 2001; Lohmann et al., 2003). Mutations at these sites have a strong impact on Con1 replication with a K1846T substitution enhancing replication to a greater extent than V1897A (Lohmann et al., 2003). In contrast to the highly adaptive mutations in NS4B and NS5A, amino acid substitutions within NS5B are only moderately enhancing. For instance, a single amino acid substitution at position 2884 (R2884G; Fig. 1B and E) increases G418-colony formation by ~500-fold compared to the parental sequence (Lohmann et al., 2001).

Another cluster of adaptive mutations (Fig. 1B) mapped to the solvent-accessible surface of the NS3 crystal structure and at a distance from the active sites of the protease and helicase (Fig. 1C and D). Interestingly, the mutations lying within NS3 were always found in conjunction with highly adaptive substitutions (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). By themselves, these NS3 mutations have minimal or no impact on replicon replication (Krieger et al., 2001; Lohmann et

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al., 2001; Lohmann et al., 2003; Lanford et al., 2003), but can enhance replication synergistically when combined with each other or with highly adaptive mutations in NS4B, NS5A, or NS5B (Krieger et al., 2001; Lohmann et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). To illustrate this point, two amino acid substitutions in NS3 (E1202G and T1280I) together with either K1846T in NS4B (Lohmann et al., 2003) or S2197P in NS5A (Krieger et al., 2001) enhance Con1 replication to levels above those observed for the replicons harboring the single NS4B or NS5A adaptive mutations. In contrast, combinations of highly adaptive mutations in NS4B, NS5A, and NS5B are antagonistic, albeit to different extents. Combining highly adaptive mutations in NS5A with each other (Blight et al., 2002b; Lohmann et al., 2003) or with the NS5B R2884G substitution (Lohmann et al., 2003) severely impair or completely abolish replication. Thus, it appears that the mechanism(s) of cell culture adaptation achieved by mutations in NS3 is different from the one exerted by substitutions in NS4B, NS5A, and NS5B.

At this stage, we can only speculate about the mechanism(s) for adaptive mutation-enhanced replication and the synergy between mutations in NS3 and those in NS4B, NS5A, or NS5B. Most mutations conferring cell culture adaptation have not been found in natural isolates of HCV and almost invariably target amino acid residues that are conserved between different HCV genotypes, suggesting that mutations represent a specific adaptation to the Huh-7 cell environment. Given that adaptive mutations reside on the surface of the available crystal structures for NS3 and NS5B (Fig. 1C-E) and do not affect the active sites of these enzymes, it is assumed that mutations modulate interactions among viral proteins and/or between viral and cellular components of the HCV replication complex. There is accumulating evidence that the suppression of NS5A hyperphosphorylation may represent a mechanism of replicon adaptation. In support of this, Con1 subgenomic RNA no longer requires adaptive mutations to efficiently replicate when Huh-7 cells are treated with inhibitors that block NS5A hyperphosphorylation (Neddermann et al., 2004). Additionally, site-directed mutagenesis of the serine residues involved in NS5A hyperphosphorylation led to a decrease in p58 formation with a corresponding increase in HCV replication (Appel et al., 2005b). Similarly, highly adaptive mutations targeting serine residues in NS5A either ablate (eg. S2204I; Blight et al., 2000) or impair NS5A hyperphosphorylation (eg. S2197P/C; Blight et al., 2000). In addition, the replication-enhancing mutations in NS4B also reduce the level of NS5A hyperphosphorylation (Evans et al., 2004b; Appel et al., 2005b). Thus, impaired NS5A hyperphosphorylation is a critical requirement for efficient Con1 RNA replication in Huh-7 cells. Perhaps hyperphosphorylation of NS5A performs a regulatory role in the HCV life cycle and adaptive mutations that suppress NS5A hyperphosphorylation prevent the dissociation of the replication complex, thereby allowing the establishment of ongoing efficient replication (Evans et al., 2004b; Appel et al., 2005b). Alternatively, it has been shown that antiviral pressures of the

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host cell triggered by HCV RNA replication contribute to the acquisition of adaptive mutations. Specifically, when a Huh-7 cell clone supporting replication of an IFN-sensitive Con1 subgenomic RNA was maintained long term in culture, mutations accumulated throughout the HCV-coding region. The fitness of this replicon variant was significantly enhanced and was resistant to the host defenses triggered by productive replication and by IFN-α treatment (Sumpter Jr. et al., 2004).

As observed for many other viruses, especially those with high mutation rates, passages in cell culture for prolonged periods of time can result in the accumulation of mutations that often improve virus replication in vitro but frequently lead to attenuation in vivo. Similarly, cell culture-adaptive mutations that facilitate efficient HCV replication in Huh-7 cells give rise to highly attenuated phenotypes in vivo. Intrahepatic inoculation of chimpanzees, the only recognized animal model for HCV infection, with full-length Con1 genomes harboring three cell culture-adaptive mutations (E1202G and T1280I in NS3 and S2197P in NS5A; Fig. 1B) failed to develop a productive infection (Bukh et al., 2002). A Con1 genome with the single S2197P substitution in NS5A replicated poorly, and one week after inoculation circulating HCV genomes were detectable but had reverted to the original wild-type Con1 sequence (Bukh et al., 2002). Thus, the attenuation in vivo of Con1 genomes carrying cell culture-acquired mutations may explain why Huh-7 cells supporting full-length Con1 replication do not produce infectious virus particles (Pietschmann et al., 2002; Blight et al., 2002b and discussed below).

GENERATION OF REPLICONS FROM OTHER HCV ISOLATESSlight variations in HCV sequence can dramatically alter the replicative ability of engineered replicons and as a result creating functional subgenomes for different HCV isolates has not been straightforward. Of the six HCV genotypes, viable replicons have only been reported for genotype 1 and 2 strains. Six genotype 1b isolates - Con1 (discussed above), HCV-N (Guo et al., 2001; Ikeda et al., 2002), HCV-BK (Grobler et al., 2003), HC-J4 (Maekawa et al., 2004), 1B-2/HCV-O (Kato et al., 2003a), and 1B-1/M1LE (Kishine et al., 2002) - productively replicate in Huh-7 cells. Replication-competent genotype 1a replicons are derived from the Hutchinson strain (H77 or HCV-H; Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004; Liang et al., 2005) and efficient replication of JFH-1, classified as a genotype 2a virus, has been also demonstrated in cell culture (Kato et al., 2003b).

GENOTYPE 1bFor many years, the only HCV replicons able to autonomously replicate in cultured Huh-7 cells were derived from the Con1 strain. This restriction has now been overcome by the development of replication-competent subgenomic RNAs derived from independent genotype 1b isolates. Unlike the Con1 replicons, cell culture

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adaptation does not appear to be required for efficient replication of subgenomes derived from the HCV-N isolate, nor for the G418 selection of Huh-7 clones (Guo et al., 2001; Ikeda et al., 2002). Instead, a unique four amino acid insertion naturally present within the ISDR of the HCV-N NS5A protein was sufficient for persistent replication in Huh-7 cells. Removal of these four amino acids from the HCV-N replicon drastically reduced its capacity to confer resistance to G418, but replication could be restored by incorporation of the highly adaptive Con1 mutation, S2204I in NS5A (Ikeda et al., 2002). Thus, this natural four amino acid insertion in HCV-N NS5A behaves like a cell culture-acquired adaptive mutation. For the HCV-BK strain, systematic mutagenesis of the NS3 coding region showed that efficient replicon replication required a mutation in the helicase domain of NS3 (R1496M) in addition to the S2204I substitution in NS5A (Grobler et al., 2003). NS5A adaptive mutations S2197P, S2204I, or ∆S2201 (Fig. 1B) were necessary for productive replication of chimeric HC-J4 replicons containing the 5' NTR and first 75 amino acids of NS3 from the Con1 strain (Maekawa et al., 2004). While the above genotype 1b replicons were derived from cDNA clones, subgenomic replicons have also been constructed from HCV genome RNA replicating at low levels in cultured cells infected with human serum containing genotype 1b HCV. A very low number of G418-resistant colonies was obtained after transfection of Huh-7 cells, or more permissive Huh-7 sublines, with subgenomic replicons derived from the human T-cell line MT-2C infected with HCV isolate M1LE (Kishine et al., 2002) or from human non-neoplastic hepatocytes (PH5CH8) infected with HCV isolate HCV-O (Kato et al., 2003a). Subgenomes replicating in G418-selected cell clones harbored mutations in NS3 as well as NS4B or NS5A. Although the adaptive advantage of these mutations for M1LE and HCV-O replication has not been determined, substitutions at these positions in the Con1 polyprotein have been shown to enhance Con1 subgenomic replication (Lohmann et al., 2001; Lohmann et al., 2003; Lanford et al., 2003).

GENOTYPE 1aThe identification of efficiently replicating replicons corresponding to genotype 1a strains has proven even more challenging than generating functional genotype 1b subgenomic RNAs. Attempts to construct a replication-competent replicon from the HCV-1 infectious clone have been unsuccessful, despite the inclusion of adaptive mutations identified in the genotype 1b Con1 replicon (Lanford et al., 2003). Similar negative results were obtained for the H77 strain until highly permissive cell lines were isolated (Blight et al., 2003; Grobler et al., 2003) or H77-Con1 chimeric replicons were constructed (Gu et al., 2003; Yi and Lemon, 2004). Although intrahepatic inoculation of H77 RNA is associated with high viremia during the acute phase of infection in the chimpanzee (Kolykhalov et al., 1997; Yanagi et al., 1997), replicons derived from this infectious H77 molecular clone require at least two adaptive mutations to productively replicate in cell culture. Interestingly, the

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single adaptive mutation S2204I in NS5A that was identified in the Con1 replicon was a crucial prerequisite for obtaining G418-resistant colonies supporting H77 replication (Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004), indicating that at least one of the Con1 adaptive mutations is also effective in a genotype 1a sequence.

Transfection of a highly permissive Huh-7 subline, Huh-7.5 (Blight et al., 2002b and see below), with S2204I-containing H77 replicons allowed the establishment of the first G418-resistant colonies supporting H77 replication (Blight et al., 2003). Analysis of H77 RNAs replicating in these G418-selected cell clones identified a second amino acid substitution in the helicase domain of NS3 (A1226D or P1496L; Fig. 2A and C). Both of these NS3 mutations, when combined individually with S2204I in NS5A, increased the replicative capacities of subgenomic H77 RNA with the greatest enhancement seen with the P1496L substitution. Similarly, Grobler et al. (Grobler et al., 2003) independently found that P1496L (or S1222T) together with S2204I is sufficient for productive replication of H77 RNA in a hyper-permissive Huh-7 subline, MR2. Like the synergistic NS3 mutations identified in the Con1 strain, H77 residues S1222, A1226, and P1496 map to the surface of the NS3 helicase and are not located in the nucleotide, metal, or nucleic acid binding sites (Fig. 2C). Additionally, replacement of proline with leucine at position 1496 has no effect on the in vitro unwinding activity of purified NS3 helicase (Grobler et al., 2003), suggesting that these mutations are involved in mediating interactions with viral or cellular factors rather than increasing the enzymatic capacity.

Alternatively, the requirements for productive H77 replication in the parental Huh-7 cell line were defined through the construction of chimeric replicons between Con1 and H77 sequences harboring S2204I in NS5A (Gu et al., 2003; Yi and Lemon, 2004). One study (Yi and Lemon, 2004) identified adaptive mutations within NS3, NS4A, and NS5A that act cooperatively to enhance H77 replication. Maximal non-chimeric H77 replication was achieved with a combination of mutations Q1067R and V1655I in NS3, K1691R in NS4A, and K2040R and S2204I in NS5A (Fig. 2A). In contrast to the NS3 mutations found in the helicase domain (Fig. 2C; Blight et al., 2003; Grobler et al., 2003), the substitutions identified in NS3 by Yi and Lemon (Yi and Lemon, 2004; Fig. 2A) are located in close proximity to the protease active site in the NS3/4A crystal structure (Q1067 and G1188; Fig. 2B) or in the P3 position of the NS3/4A cleavage site potentially influencing substrate recognition during cis-cleavage at this junction (V1655; Fig. 2A). Furthermore, K1691 in NS4A is located immediately downstream of the sequence involved in complex formation with NS3. Thus, these mutations (Yi and Lemon, 2004) may facilitate HCV replication via a mechanism different from those amino acid substitutions previously identified in the helicase domain of NS3 (Blight et al., 2003; Grobler et al., 2003). Similarly, another laboratory (Gu et al., 2003) achieved efficient replication of a replicon that

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was predominantly H77 derived, except the 5' NTR and N-terminal 75 amino acids of NS3 were from the Con1 genotype 1b strain. In the single G418-resistant cell clone analyzed, four amino acid changes were identified across NS3, NS5A, and NS5B; however, it is unclear which mutations or combination of mutations was responsible for augmenting chimeric or non-hybrid replication (Gu et al., 2003).

Fig. 2. Location of adaptive mutations that enhance H77 replicon replication in Huh-7 cells. (A) The NS3 to NS5B polyprotein with the positions of individual mutations proven to facilitate efficient replication in combination with S2204I in NS5A (bold) shown on the right. The protease (P) and helicase (H) domains in NS3 and the ISDR in NS5A are illustrated. Solvent-accessible surface of the NS3/4A protease (B) and NS3 helicase (C) crystal structures. The adaptive mutations and other significant features are highlighted as described in Fig. 1. The coordinates of these structures were retrieved from protein database under accession number 1A1R (Kim et al., 1996) and 1A1V (Kim et al., 1998) for the models shown in B and C, respectively. This figure can be viewed in color at http://blightlab.wustl.edu/chapter11/.

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In complete contrast, an H77-derived replicon encoding the puromycin N-acetyltransferase (PAC) gene instead of the Neo cassette (Fig. 3A) does not require adaptive mutations to autonomously replicate and confer resistance to puromycin in Huh-7 cells, although S2204I in NS5A did evolve in a minority of cell clones (Liang et al., 2005). Additionally, unlike the studies discussed above (Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004), inclusion of S2204I in NS5A did not significantly improve the colony-forming efficiency of PAC-expressing replicons (~3-fold improvement; Liang et al., 2005). The ability of PAC-expressing H77 replicons to establish replication in Huh-7 cells without adaptive mutations is puzzling and may reflect the different selective pressure. Perhaps cellular environments that are conducive to non-adapted H77 replication are more efficiently selected by puromycin than G418. In support of this, transfection of total cellular RNA extracted from a replicon-containing cell clone led to a 50-fold increase in puromycin-resistant colonies, suggesting that translation of cellular mRNAs initially introduced with replicon RNA created an intracellular milieu in naïve Huh-7 cells favorable for the establishment of HCV replication.

GENOTYPE 2aSubgenomic replicons derived from the genotype 2a JFH-1 clone, initially isolated from a patient with fulminant hepatitis, represent the only non-genotype 1 sequence currently capable of efficient replication in cell culture (Kato et al., 2003b). Interestingly, JFH-1 subgenomes replicate with high efficiency in Huh-7 cells in the absence of adaptive mutations; the G418-resistant colony-forming ability of the unmodified bicistronic replicon is 60-fold higher than a Con1 subgenomic RNA harboring highly adaptive mutations (Kato et al., 2003b). Although adaptive mutations are not a prerequisite for efficient JFH-1 replication, amino acid changes in the replicase proteins were identified in the majority of G418-selected replicon-containing Huh-7 clones. Of those tested, one mutation, H2476L in NS5B, enhanced the G418 transduction efficiency by only 3-fold, which is well below the level of enhancement seen for single highly adaptive Con1 mutations (Blight et al., 2000; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). Nonetheless, the colonies derived from JFH-1 replicons containing this NS5B mutation were significantly larger than those obtained after transfection of unmodified JFH-1 subgenomes (Kato et al., 2003b), suggesting that this mutation confers a higher replication phenotype in Huh-7 cells. Furthermore, the high replication efficiency of unmodified JFH-1 replicons allowed HCV RNA and proteins to be monitored in transient replication assays (Fig. 4). Based on the data presented by Kato and coworkers (Kato et al., 2003b), the JFH-1 subgenomic RNA is the most efficient replicon tested so far. Additionally, it appears that adaptive mutations may not always be necessary for efficient replication in cell culture. Instead, the requirement for adaptive mutations is dependent on the individual HCV isolate. Identification of the JFH-1 determinants that promote this high level of RNA replication could provide

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Fig. 3. Organization of replication-competent HCV replicons reported so far. (A) Antibiotic selectable replicons. (Top) Biscistonic HCV replicons are composed of the 5' NTR, a small portion of the capsid-coding region (open box), an antibiotic resistance gene (neomycin phosphotransferase [Neo], blasticidin S deaminase [blast], hygromycin phosphotransferase [hygro], or puromycin acetyltransferase [PAC]; black box), the EMCV IRES (EMCV; solid line), the NS3-5B coding region and the 3' NTR. (Bottom) Monocistronic hygromycin selectable replicon consisting of the 5' NTR, the first 16 amino acids of the capsid fused to the hygromycin phosphotransferase gene (hygro; black box), ubiquitin coding sequence (Ub), the HCV non-structural proteins NS3 to NS5B (open box), and the 3' NTR. (B) Replicons encoding reporter genes or a transactivator of SEAP expression. The 5' and 3'

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insights into the mechanisms of HCV RNA replication. Replication of a genotype that diverges from genotype 1 isolates by ~30% not only represents an important advance for replication studies, but also for drug discovery efforts.

DEVELOPMENT OF ALTERNATIVE REPLICON DERIVATIVESThe identification of adaptive mutations that dramatically enhance genotype 1 HCV replication in cell culture has made it possible to explore alternative drug resistance genes (Fig. 3A; Frese et al., 2002; Evans et al., 2004a; Liang et al., 2005; Appel et al., 2005a) and develop transient replication assays utilizing replicon derivatives expressing or activating the expression of easily quantifiable reporter enzymes (Fig. 3B; Krieger et al., 2001; Yi et al., 2002; Lohmann et al., 2003; Murray et al., 2003; Ikeda et al., 2005). Furthermore, robustly replicating monocistronic replicons, containing one translation module and eliminating non-HCV sequences (Fig. 3C; Blight et al., 2003), have also been generated, as well as replication-competent full-length RNAs that stably or transiently replicate in Huh-7 cells (Fig. 3D; Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Yi and Lemon, 2004; Ikeda et al., 2005).

DRUG RESISTANCE GENESEngineering alternative drug resistance genes may permit faster selection of replication-positive cells as well as allow different replicon sequences to be sequentially or simultaneously selected within the same cell. Neo-encoding Con1 replicons have been successfully selected in the same cell with Con1 bicistronic replicons encoding either blasticidin S deaminase (Evans et al., 2004a) or hygromycin phosphotransferase (Appel et al., 2005a) genes in place of the Neo cassette (Fig. 3A). This approach found: (i) no evidence of recombination between different subgenomes (Evans et al., 2004a; Appel et al., 2005a); (ii) a high level of competition between subgenomic RNAs, suggesting that host cell machinery required for HCV replication is limiting (Evans et al., 2004a) and; (iii) replicons harboring lethal mutations in NS3, NS4B, or NS5B or mutations that disrupt the structure of the N-terminal membrane anchor of NS5A could not be rescued by co-expression of functional subgenomic RNAs (Evans et al., 2004a; Appel et al., 2005a). Thus, it appears that HCV replication complexes are relatively closed

NTR structures are shown, the first 12 amino acids of capsid and the HCV ORF are depicted by open boxes and the reporter genes, firefly luciferase (fLUC), β-lactamase (bla), GFP, and renilla luciferase (rLUC) are represented by hatched boxes. The Neo gene (Neo; black box), the EMCV IRES (EMCV; solid line), the poliovirus IRES (Polio; dashed line), ubiquitin (Ub; shaded box), the HIV tat protein (tat; hatched box), and the foot and mouth disease virus protease 2A (2A; shaded box) are depicted. The curved arrow indicates the site of autocatalytic 2A-mediated cleavage. (C) Monocistronic HCV subgenome containing the 5' NTR, the capsid (C)-coding region fused to the NS2-5B ORF (open box) followed by the 3' NTR. (D) Full-length HCV replicons encoding the entire HCV polyprotein coding sequence (C-NS5B; open box). The 5' and 3' NTRs, the Neo gene (Neo; black box), the renilla luciferase gene (rLUC; hatched box) and EMCV IRES (EMCV; solid line) are illustrated.

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structures preventing or limiting the exchange of viral proteins. However, deletions in NS5A or multiple mutations affecting potential phosphorylation sites in the central region of NS5A could be complemented in trans, suggesting that NS5A is loosely associated with the intracellular membranes that provide the scaffold of the HCV replication complex (Appel et al., 2005a). Alternatively, a monocistronic replicon encoding the hygromycin phosphotransferase gene establishes productive replication in Huh-7 cells and confers resistance to hygromycin (Frese et al., 2002). In this replicon, ubiquitin was inserted in-frame between hygromycin phosphotransferase and the NS3-5B coding region so that translation of the entire ORF is directed by the HCV 5' NTR and NS3 is released from the polyprotein by a cellular ubiquitin carboxyl-terminal hydrolase-mediated cleavage event at the ubiquitin/NS3 junction (Fig. 3A).

REPORTER GENESAssays for colony formation are time consuming and assume that the frequency of drug-resistant colonies observed with a given replicon is directly proportional to its intrinsic replication activity. With the identification of adaptive mutations that facilitate efficient HCV replication, transient RNA replication assays that allow a more rapid and direct analysis of relative replication efficiencies have been developed. Reporters such as luciferase and ß-lactamase, as well as a transactivator inducing secreted alkaline phosphatase (SEAP), have been used to monitor replication at early times after transfection of Huh-7 cells.

Firefly luciferase has successfully replaced the Neo gene in Con1 (Krieger et al., 2001) and HCV-O (Ikeda et al., 2005) bicistronic replicons (Fig. 3B), thus enabling replication to be monitored at various times following transfection by measuring the luciferase activity relative to a polymerase-defective replicon. After 48-72 hours, the luciferase activities seen with an adapted Con1 replicon are about 100-fold higher than the negative control (Krieger et al., 2001). The luciferase activity directly correlates with the levels of HCV RNA synthesis, demonstrating that luciferase is a reliable marker of replication (Krieger et al., 2001). Enhanced replication levels, and thus higher luciferase activities (~5-fold), as well as more reproducible results were achieved by placing luciferase under the translational control of the IRES from poliovirus instead of the HCV IRES (Fig. 3B; Lohmann et al., 2003). Although luciferase activity allows the rapid determination of relative replication levels in the population of transfected cells, it does not provide a direct measure of the number of cells supporting replication. This restriction has been overcome by the development of bicistronic Con1 replicons containing a ß-lactamase reporter (Fig. 3B; Murray et al., 2003). In this strategy, Huh-7 cells supporting active HCV replication are identified using a cell-permeable fluorescent substrate that is cleaved by ß-lactamase expressed in the cell, leading to blue fluorescence. More recently, it has been reported that the C-terminal domain of NS5A (between residues 2370 and

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2412) is dispensable for replicon function (Appel et al., 2005b) and heterologous sequences can be inserted within this region with only a moderate reduction in replication (Moradpour et al., 2004b; Appel et al., 2005b). Thus, viable Con1 replicons carrying an in-frame insertion of enhanced green fluorescent protein (GFP) at position 2356 or 2390 in NS5A were created (Fig. 3B); however, the G418 transduction efficiency of GFP-expressing replicons was ≥25-fold lower than the parental replicon constructs (Moradpour et al., 2004b). Although the GFP signal is readily visualized in G418-selected cell clones by fluorescence microscopy allowing active replication complexes to be tracked in real time (Moradpour et al., 2004b and see below), it is not clear if the replication competence of these GFP-expressing replicons is sufficient for the detection and quantification of GFP-positive cells in transient replication assays. In an independent report a firefly luciferase-expressing Con1 subgenomic RNA (Fig. 3B) carrying a GFP insertion between positions 2370 and 2412 of NS5A replicated to levels about 100-fold below the parental replicon that lacked GFP and this lower replication efficiency prevented the direct visualization of the NS5A-GFP fusion protein at 72 hr post-transfection (Appel et al., 2005b).

The first cistron of the bicistronic Con1, HCV-N, (Yi et al., 2002) and H77 (Yi and Lemon, 2004) replicons have been modified to include the human immunodeficiency virus (HIV) tat protein, a potent transcriptional transactivator of the HIV long terminal repeat (LTR) promoter (Yi et al., 2002). Briefly, the tat-coding sequence was fused to a picornaviral 2A protease sequence followed by the Neo selectable marker (Fig. 3B), such that upon translation, the autocatalytic protease activity of 2A mediates cleavage at its C-terminus, liberating Neo. Replication in transfected cells leads to the intracellular accumulation of tat, which in turn activates the LTR-SEAP cassette, which is stably integrated into the genome of the transfected Huh-7 cell. SEAP is subsequently expressed and secreted from replication-positive cells and five days after transfection of adapted replicons, extracellular SEAP can reach levels 100-fold above the amount secreted from cells transfected with replication-defective mutants (Yi et al., 2002).

In addition to the use of these reporter gene replicon systems for rapid determination of replicative ability, these systems are amenable to high throughput tests including screening large compound libraries for anti-HCV activity. For instance, the ß-lactamase replicon system has been successfully adapted to a high-throughput screening assay to identify inhibitors of HCV replication (Zuck et al., 2004). Furthermore, replicons carrying firefly or renilla luciferase fused to the neomycin phosphotransferase gene (Fig. 3B) have been used to assess the effect of human IFN-α and ribavirin on HCV replication (Tanabe et al., 2004; Ikeda et al., 2005). Another group determined the level of biologically active IFN-α in sera taken from HCV carriers undergoing IFN treatment by using cells harboring a bicistronic

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replicon where the first cistron comprises a firefly luciferase-ubiquitin-Neo cassette (Fig. 3B; Vrolijk et al., 2003).

MONOCISTRONIC SUBGENOMIC REPLICONSThe ability to monitor HCV replication without selection eliminates the requirement for bicistronic replicons. In fact, heterologous sequences such as the Neo gene and the EMCV IRES reduce the replicative capacity of Con1- and H77-derived subgenomic RNAs (Blight et al., 2002b; Blight et al., 2003). Robust replication is observed with a monocistronic replicon composed of the 5' NTR followed by the entire capsid sequence fused to the NS2-NS5B coding region, such that cleavage between capsid and NS2 is mediated by the host cell signal peptidase and translation is under the control of the HCV IRES (Fig. 3C; Blight et al., 2003). The replication-competence of this subgenomic RNA, as well as the monocistronic hygromycin phosphotransferase-encoding replicon described above (Fig. 3A; Frese et al., 2002), demonstrates that the EMCV IRES is not a requirement for efficient expression of the HCV replicase region and subgenomic replication in Huh-7 cells. Since this monocistronic replicon lacks antibiotic resistance or reporter genes (Fig. 3C), replication in transfected cells can only be monitored by the detection of HCV-specific RNA and proteins. Quantitative real-time RT-PCR (Blight et al., 2000; Blight et al., 2002b; Blight et al., 2003) and Northern blot hybridization (Krieger et al., 2001; Pietschmann et al., 2002; Lohmann et al., 2003; Kato et al., 2003b; Yi and Lemon, 2004) have been used to analyze HCV RNA synthesis (Fig. 4), and HCV protein expression has been successfully detected by a collection of assays including Western blot, fluorescent activated cell sorting (FACS), metabolic labeling and immunoprecipitation with HCV-specific antisera, and immunostaining of cell monolayers (Fig. 4; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Kato et al., 2003b). HCV subgenomic replication can now be studied in the absence of heterologous sequences, alleviating concerns that the experimental phenotype is related to the expressed heterologous gene or foreign IRES element.

FULL-LENGTH REPLICONSFull-length or genomic HCV replicons derived from Con1, H77, HCV-O, and HCV-N productively replicate in Huh-7 cells or in highly permissive Huh-7 sublines (Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Yi and Lemon, 2004; Ikeda et al., 2005). Full-length HCV RNAs carry the complete HCV open reading frame (C-NS5B) and replication is dependent on cell culture-adaptive mutations. Bicistronic derivatives encoding Neo (Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Ikeda et al., 2005) or a renilla luciferase-Neo fusion (Ikeda et al., 2005) have been generated (Fig. 3D), facilitating the selection of stable G418-resistant cell lines supporting full-length HCV replication. The number of Huh-7 cells able to support full-length replication is much lower than that seen for the subgenomic derivative carrying the same adaptive mutations and the average level of full-length RNA replication is about

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Fig. 4. The intracellular steps involved in HCV RNA replication and the various methods used to monitor these steps in stable antibiotic-resistant cell clones or in transient replication assays. After transfection of Huh-7 cells with in vitro transcribed RNAs (lightening bolt), measurable reporters, transactivators inducing reporter gene expression, antibiotic resistance markers, and the HCV polyprotein are expressed via IRES-dependent translation (A). (B) The HCV polyprotein is processed into the individual proteins and the NS3-5B proteins associate to form a membrane-associated replication complex. (C) Positive-sense HCV RNA is transcribed into a complementary negative-sense intermediate and progeny positive-sense HCV RNAs then serve as templates for additional negative-sense RNA synthesis (small open arrows) or further translation (large open arrow). Upon establishment of HCV replication, foreign genes are expressed to levels allowing antibiotic selection and/or quantification of reporter gene expression (A). HCV protein production (B) and RNA synthesis (total, positive- or negative-sense HCV RNA; C) can be measured by the methods shown. Abbreviations: IP – immunoprecipitation; actD – actinomycin D; FACS – fluorescent activated cell sorting.

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5-fold lower than subgenomic replication (Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003). So far, there is no evidence of HCV particle assembly and release from Huh-7 cells supporting replication of Con1 (Pietschmann et al., 2002; Blight et al., 2002b) or H77 (Blight et al., 2003) full-length RNAs containing cell culture-adaptive mutations. Although unmodified full-length HCV RNAs generated from these HCV strains produce infectious virus in the chimpanzee model (Kolykhalov et al., 1997; Yanagi et al., 1997; Bukh et al., 2002), Con1 genomes harboring cell culture-adaptive mutations are severely attenuated in vivo (Bukh et al., 2002), suggesting that adaptive mutations inhibit virus particle assembly. In support of this, Huh-7 cells supporting replication of a full-length RNA containing the non-structural proteins from the genotype 2a JFH-1 strain that lacks adaptive mutations assemble and release infectious virus particles (Bartenschlager et al., 2004; Heller et al., 2005). Additionally, virus production has also been observed in Huh-7 cells transfected with plasmid DNA carrying an unmodified infectious full-length genotype 1b HCV genome flanked by self-cleaving hammerhead ribozymes to generate the exact 5' and 3' ends of intracellular transcribed RNA (Heller et al., 2005). The development of cell culture systems supporting the complete virus life cycle now allows studies directed towards defining the mechanisms of viral particle assembly (see Chapter 16).

CELL LINES PERMISSIVE FOR HCV REPLICATIONIn vivo, hepatocytes are believed to be the major site of HCV replication; however, some evidence suggests that extrahepatic cells, including lymphocytes, monocytes, and dendritic cells, may also harbor the virus (Blight and Gowans, 1995; Laskus et al., 2000; Goutagny et al., 2003). Productive replication of HCV replicons in vitro appears to be extremely cell-type specific, with human hepatoma Huh-7 cells being the most permissive cell line identified so far, although, as described below, the environment within these cells affects the efficiency of HCV replication. Extensive effort has been devoted to identifying other permissive cell lines and recently, the cell repertoire was expanded to include additional continuous human hepatoma cell lines and a murine hepatoma cell line, as well as non-hepatic cell lines such as the human cervical cancer-derived HeLa cell line and 293 cells established from human embryonic kidney.

Huh-7 CELL LINEAlthough cell culture-adaptive mutations are essential for genotype 1 replication in Huh-7 cells, there is convincing evidence that the environment within the Huh-7 cell also governs the ability of HCV RNAs to replicate. First, the relative replication efficiencies of subgenomic RNAs in transient replication assays can vary by as much as 100-fold between different passages of Huh-7 cells. These differences are independent of the adaptive mutation(s) introduced into the replicon and are not due to differences in HCV RNA translation or stability (Lohmann et al., 2003),

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suggesting that efficient replication depends on host cell conditions or specific cellular factors. Secondly, replication of subgenomic RNAs, despite the inclusion of highly adaptive mutations, can only be detected in a subset of transfected Huh-7 cells (~10% for the highly adaptive mutation S2204I in NS5A; Blight et al., 2000; Blight et al., 2002b), suggesting that many Huh-7 cells do not provide an optimal environment for HCV replication. Additionally, the number of G418-resistant colonies obtained after transfection of selectable replicons was significantly higher in cell clones from which the replicon had been eliminated by extended treatment with IFN-α ("curing") than that observed for naïve Huh-7 cells (Blight et al., 2002b; Murray et al., 2003; Ikeda et al., 2005). Furthermore, rare G418-selected Huh-7 clones "cured" of Con1 replicons that had not acquired adaptive mutations during the selection process were more permissive for HCV replication than "cured" cells originally supporting adapted Con1 subgenomic RNA replication (Blight et al., 2002b). These results demonstrate that cellular environments more conducive to HCV replication are ultimately selected and, in Huh-7 cells that harbor conditions most supportive for viral replication, transfected Con1 replicon RNA does not need to adapt to efficiently replicate.

The most permissive "cured" subline identified so far (Huh-7.5; Blight et al., 2002b) has the capacity to support high levels of subgenomic HCV replication in >75% of transfected cells. Furthermore, Huh-7.5 cells more readily support RNAs with lower replicative abilities, such as full-length Con1 replicons (Blight et al., 2002b) and H77-derived RNAs (Blight et al., 2003). Increased permissiveness in Huh-7.5 cells is due to mutational inactivation of the retinoic acid inducible gene-I (RIG-I), a cytoplasmic protein that recognizes structured RNA to induce type I IFN production via activation of transcription factors interferon regulatory factor (IRF)-3 and NF-κΒ. Complementation with functional RIG-I restores IRF-3 signaling in Huh-7.5 cells and converts this hyper-permissive cell line to a relatively non-permissive phenotype (Sumpter Jr. et al., 2005). Thus, RIG-I-mediated activation of IRF-3 is a critical determinant of cellular permissiveness for HCV replication.

ADDITIONAL CELL LINESAttempts by many investigators to propagate HCV RNAs in other cell lines were unsuccessful until 2003, when Zhu and coworkers described replication of Con1 subgenomic RNAs in HeLa cells and in the murine hepatoma cell line Hepa1-6, albeit with low efficiency (Zhu et al., 2003). Due to the high error rate of the NS5B RdRp, replicon RNAs prepared from Huh-7 cell lines in which persistent replication had been established were expected to have greater genetic variance than HCV RNAs generated by in vitro transcription from cloned cDNA templates. Indeed, transfection of HeLa cells with total RNA from Con1 replicon-harboring Huh-7 cells gave rise to a low number of G418-resistant cell colonies. Replicons in G418-resistant HeLa cells maintained the Huh-7 cell adaptive mutations, but

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acquired several additional mutations in the non-structural proteins. Interestingly, the colony-forming efficiency of replicon RNAs isolated from HeLa cells was significantly higher in naïve HeLa cells than Huh-7 cells. In addition, HeLa cell-derived replicon RNAs were more efficient at establishing G418-resistance in HeLa cells than replicons obtained from Huh-7 cells. These results indicate that replicon variants have been selected that can replicate more efficiently in HeLa cells. However, in vitro transcribed subgenomic RNAs carrying both the Huh-7 and HeLa cell-specific mutations only conferred G418 resistance in a few HeLa cells, and thus the relative contribution of these mutations to productive replication in HeLa cells has not been determined. Replicon RNAs derived from HeLa cells, but not from Huh-7 cells, were also able to replicate and confer resistance to G418 in a few Hepa1-6 cells. Sequence analysis revealed that replicons from these mouse cells had preserved the majority of the mutations found in HeLa cells, and only a few additional mutations were identified. Moreover, total RNA isolated from one of the Hepa1-6 replicon-containing cell lines did not increase the colony-forming efficiency compared to replicon RNA from HeLa cells, suggesting that HeLa-derived subgenomes were already adapted for replication in the mouse cells (Zhu et al., 2003).

Con1 replicon replication has also been established in human embryonic kidney 293 cells. By co-culturing replicon-containing Huh-7 cells with 293 cells, rare hybrids closely resembling parental 293 cells were selected that supported subgenomic RNA replication (Ali et al., 2004). Nucleotide sequence analysis of replicating HCV RNA in hybrid 293 cells identified a large number of mutations that appear to facilitate replication in 293 cells; transfection of total cellular RNA isolated from one of these replicon-expressing hybrid clones was able to establish replication in naïve 293 cells. As observed by Zhu and coworkers (Zhu et al., 2003), in vitro transcribed replicon RNA, containing all the mutations identified in this hybrid 293 clone, failed to confer resistance to G418 in naïve 293 cells (Ali et al., 2004). The differences in colony-forming capacity between in vitro transcribed Con1 subgenomic RNA and replicon RNA isolated from stable cell clones remains a mystery, although it is possible that RNA molecules co-purifying with the replicating replicon RNA facilitates the establishment of Con1 replication in these less permissive cell lines.

Although in vitro transcripts derived from genotype 1b replicons have not successfully established replication in non-Huh-7 cells (Zhu et al., 2003; Ali et al., 2004), G418 resistance in human hepatocyte- (HepG2 and IMY-N9; Date et al., 2004) and non-hepatocyte- (HeLa and 293; Kato et al., 2005) derived cell lines has been achieved by the genotype 2a replicon, JFH-1. The efficiency of colony formation was 10- to 1000-fold lower compared to Huh-7 cells (Date et al., 2004; Kato et al., 2005), and the colony-forming efficiency and colony size in HepG2 and IMY-N9 cells was increased following transfection of JFH-1 replicons harboring

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the Huh-7 specific adaptive mutation in NS5B (H2496L; Date et al., 2004). Many G418-selected cell clones did not contain mutations in the HCV coding region and, when coding changes were found, they were not shared between independent clones and had not been previously identified in JFH-1 replicons replicating in Huh-7 cells (Date et al., 2004; Kato et al., 2005). Interestingly, in eight of the nine 293-derived cell clones analyzed, amino acid substitutions were not identified (Kato et al., 2005). These findings suggest that adaptive mutations are not essential for JFH-1 replication in either hepatocyte- or non-hepatocyte-derived cell lines.

Collectively, the cell tropism for HCV genotypes 1b and 2a has not only been expanded to include additional human hepatoma cell lines, but also non-liver derived human cells and murine hepatocytes, disproving the previous hypothesis that HCV replication is governed by hepatocyte- and primate-specific factors. Moreover, the ability of HCV replicons to replicate in a murine hepatoma cell line offers some hope that a mouse model for HCV infection may be developed in the future.

APPLICATIONS OF THE HCV REPLICON SYSTEMSince the introduction of HCV replicons in 1999 and the subsequent identification of adaptive mutations a year later, numerous researchers have utilized the replicon system to probe the mechanisms of HCV replication, define the roles of individual proteins, identify the viral and cellular determinants of HCV replication, and examine the interplay between HCV and the Huh-7 cell. This section provides specific examples to highlight the utility of this cell culture model to address these fundamental questions about HCV biology.

The availability of stable cell lines that harbor autonomously replicating subgenomic RNAs has facilitated the study of viral protein expression, subcellular localization of HCV replication, and the structure, function, and biochemical properties of the replication complex. Additionally, the mechanisms by which HCV counteracts the host antiviral response, the effects of HCV replication on host cell function and the importance of host factors for efficient replication have begun to be unraveled. The HCV polyprotein is proteolytically processed in a preferential order with rapid cleavages at the NS3/4A and NS5A/5B sites, while the NS4A-4B-5A precursor is processed at a slower rate (Pietschmann et al., 2001), confirming previous studies using heterologous expression systems (Lin et al., 1994; Bartenschlager et al., 1994; Tanji et al., 1994). The mature HCV proteins have half-lives ranging from 10 to 16 hours, except the hyperphosphorylated form of NS5A, which appears less stable (Pietschmann et al., 2001). Similar to all positive-sense RNA viruses investigated so far (reviewed in Ahlquist et al., 2003; Salonen et al., 2005), HCV reorganizes intracellular membranes to form a membranous web. This altered membrane represents a site of HCV replication in Huh-7 cells (Gosert et al., 2003), and by utilizing the replicon encoding the NS5A-GFP fusion (Fig. 3B), active HCV

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replication complexes have been directly visualized in living cells by fluorescence microscopy (Moradpour et al., 2004b). Interestingly, fluorescence is strongest in subconfluent cells, consistent with earlier studies showing that subgenomic RNA replication is strongly influenced by the proliferation status of the cells with replication stimulated in the S phase of the cell cycle, but rapidly declining in confluent or serum-starved cells (Pietschmann et al., 2001; Scholle et al., 2004). The ability to track functional replication complexes should aid in defining the steps involved in membranous web formation and in the assembly and turnover of replication complexes. Although the origin of the membranous web has not been defined, its close proximity to the ER and the ER localization of the non-structural proteins in replicon-containing cells (Pietschmann et al., 2001; Mottola et al., 2002; El-Hage and Luo, 2003; Miyanari et al., 2003) suggest the web is derived from membranes of the ER. In contrast, the association of HCV RNA and non-structural proteins with NP40-insoluble membranes in replicon-containing cells and their cofractionation with caveolin-2 suggest that active replication complexes reside on lipid rafts recruited from intracellular sites (Shi et al., 2003; Gao et al., 2004; Aizaki et al., 2004). Clearly more experimentation is required to define the origin of the membranes on which the HCV replication complex assembles.

HCV subgenomic replicon-containing Huh-7 cells also provide a source of membrane fractions containing crude replication complexes for biochemical studies (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). These complexes retain enzymatic activity as evidenced by the synthesis of replicon-length RNA from the endogenous (co-fractionating) template RNA. Furthermore, partially single-stranded and double-stranded replicative forms are synthesized, transcription of single- and double-stranded RNAs are differentially effected by Mg2+ and Mn2+, RNA synthesis is actinomycin D-resistant, and de novo initiation of RNA transcription occurs in these isolated membrane fractions. Thus, the use of enzymatically active HCV replication complexes rather than employing NS5B alone offers an in vitro system to probe the structure and function of the replicase and to evaluate potential inhibitors targeting RNA replication. Interestingly, these crude replication complexes have failed to utilize exogenously added template RNA (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) and most of the viral RNA in these membrane-bound complexes is nuclease resistant (El-Hage and Luo, 2003; Miyanari et al., 2003; Yang et al., 2004), consistent with the notion that HCV replication complexes are relatively closed structures. Furthermore, treatment of these complexes with proteinase K degrades more than 90% of the viral proteins with no effect on RNA transcription (Miyanari et al., 2003), thus only a minor fraction of HCV proteins are engaged in RNA synthesis and this fraction is protected within the replication complex.

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Like many viral infections, HCV triggers the host cell antiviral response in part through the accumulation of replication intermediates or the presence of double-stranded RNA structures within the HCV genome (Pflugheber et al., 2002; Wang et al., 2003; Sumpter Jr. et al., 2005). Persistent HCV infections frequently develop (Alter and Seeff, 2000), suggesting that HCV has evolved efficient mechanisms to counteract the intracellular antiviral response. These mechanisms are beginning to be elucidated using stable cell lines supporting persistent subgenomic HCV replication. The protease action of the HCV NS3/4A complex has been shown to disrupt two independent signaling pathways, toll-like receptor 3 (TLR3) and RIG-I, that both induce type I IFN production (Li et al., 2005; Sumpter Jr. et al., 2005; Breiman et al., 2005). While the protease target in the RIG-I signaling pathway has yet to be identified (Sumpter Jr. et al., 2005; Breiman et al., 2005), the adaptor protein (toll-IL-1 receptor domain-containing adaptor inducing IFN-β; TRIF) linking TLR3 to the kinases responsible for activating the latent transcription factors, IRF-3 and NF-κB, is proteolytically cleaved (Li et al., 2005). Additionally, NS5A has been implicated in the ability of HCV to block the host response to double-stranded RNA. Direct binding of NS5A with protein kinase R (PKR) disrupts signaling events that activate IRF-1 (Pflugheber et al., 2002) or limit RNA translation (Wang et al., 2003).

Inhibition of host factors either through RNA interference or expression of dominant-negative mutants of the protein has facilitated the identification of host cell factors important for productive HCV replication. For example, by these strategies polypyrimidine tract binding protein (PTB; Zhang et al., 2004; Domitrovich et al., 2005), La autoantigen (Zhang et al., 2004; Domitrovich et al., 2005), and human vesicle-associated membrane transport protein A (hVAP-A; Gao et al., 2004; Zhang et al., 2004) have been found to be important for HCV replication. Furthermore, hVAP-A interacts with NS5B (Gao et al., 2004) and NS5A (Evans et al., 2004b; Gao et al., 2004) and recently it has been suggested that NS5A hyperphosphorylation disrupts interactions between hVAP-A and NS5A to negatively regulate HCV replication (Evans et al., 2004b).

Reverse genetics in the replicon system has become an important tool to define HCV RNA sequences and protein determinants critical for productive RNA replication in Huh-7 cells. For instance, the first 125 nucleotides of the 5' NTR are sufficient for RNA replication, demonstrating that the regions required for translation and replication overlap (Friebe et al., 2001; Kim et al., 2002; Reusken et al., 2003; Luo et al., 2003). Mapping studies have been conducted on the 3' NTR and confirm the observations made in chimpanzees experimentally inoculated with RNA transcripts carrying similar mutations in the 3' NTR (Kolykhalov et al., 2000; Yanagi et al., 1999). The terminal 98 nucleotides and poly(U/UC) tract are indispensable for replication, although the poly(U/UC) region can be shortened without affecting HCV replication (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003). In contrast, the upstream variable region can be deleted, resulting in a 100-fold reduction in

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G418-resistant colonies (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003). More recently, critical cis-acting replication sequences in addition to the 5' and 3' NTRs have been identified. Mutational disruption of a computer-predicted highly conserved stem-loop structure located within the 3' terminal coding region of NS5B, designated 5BSL3.2, blocks subgenomic replication (Lee et al., 2004a; You et al., 2004; Friebe et al., 2005) and can be restored when an intact copy of this RNA element is inserted into the 3' NTR (Friebe et al., 2005). Furthermore, 5BSL3.2 and the terminal 98 nucleotides form a pseudoknot structure that is indispensable for HCV replication (Friebe et al., 2005).

As mentioned above, HCV replication occurs in conjunction with rearranged membranes, and thus it is not surprising that most of the HCV non-structural proteins contain membrane-anchoring segments (NS4A, 4B, 5A, and 5B; reviewed in Dubuisson et al., 2002; Moradpour et al., 2003). Membrane association of NS5A and NS5B, mediated by an N-terminal amphipathic helix in NS5A (Elazar et al., 2003) and the C-terminal 21 amino acid residues of NS5B (Moradpour et al., 2004a; Lee et al., 2004b), is essential for productive HCV replication in Huh-7 cells. Although the determinants for membrane association have been defined and their importance in HCV replication is beginning to be recognized, the composition and interactions required to assemble functional replication complexes are poorly understood. Nonetheless, sequences that may mediate interactions essential for replicase assembly are being identified. For instance, solvent-exposed residues in the N-terminal helix of NS5A (Penin et al., 2004) and sequences within the transmembrane region of NS5B (Ivashkina et al., 2002; Moradpour et al., 2004a; Lee et al., 2004b) appear to play critical, but as yet undefined, roles in subgenomic replication beyond those of membrane insertion. Two regions of NS5A previously reported to mediate interactions with NS5B in vitro (Shirota et al., 2002) have been shown to be important for productive HCV replication (Shimakami et al., 2004).

Finally, by applying reverse genetics in the replicon system, a GTP-binding motif (Einav et al., 2004) and an amphipathic helix (Elazar et al., 2004) in NS4B as well as a conserved zinc-binding motif in the N-terminal domain of NS5A (Tellinghuisen et al., 2004) have been found to be important for efficient HCV replication, although at this stage the exact role of these motifs in replication has not been determined. As discussed above, hyperphosphorylated NS5A is detrimental to HCV replication, while basal phosphorylation of NS5A has recently been shown to be nonessential for productive HCV replication in Huh-7 cells (Appel et al., 2005b). Thus, further experimentation is warranted to determine whether phosphorylation of NS5A is required at all for RNA replication or whether it serves a regulatory purpose, such as signaling the switch between RNA replication and virus particle assembly. As illustrated above, subgenomic replicons provide an excellent model system for examining HCV-host interactions and for molecular studies of HCV replication.

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INHIBITION OF REPLICON REPLICATIONWith the development of HCV replicons it became possible to analyze the role of cytokines in the cellular defense against HCV. Genotype 1 replication in cell culture is sensitive to the antiviral programs induced by IFN-α (Blight et al., 2000; Frese et al., 2001; Guo et al., 2001; Gu et al., 2003; Kato et al., 2003a; Lanford et al., 2003; Tanabe et al., 2004; Kanda et al., 2004; Ikeda et al., 2005), IFN-β (Kato et al., 2003a; Larkin et al., 2003), IFN-γ (Frese et al., 2002; Kato et al., 2003a; Lanford et al., 2003; Larkin et al., 2003), IFN-λ (Robek et al., 2005), and interleukin-1 (IL-1; Zhu and Liu, 2003), but not tumor necrosis factor-α (Lanford et al., 2003). In all cases examined so far, the 50% inhibitory concentrations (IC50) for IFN-α in Huh-7 cells are very low (0.5-3 IU/ml; Gu et al., 2003; Tanabe et al., 2004; Ikeda et al., 2005) and are independent of the ISDR (Blight et al., 2000). Type I IFNs induce the transcription of a large number of genes that encode effector proteins with antiviral activities, including PKR, 2'-5' oligoadenylate synthase (OAS), IFN-stimulated gene 56 (ISG56), and MxA guanosine triphosphatase. Although MxA inhibits the replication of a broad variety of RNA viruses (Haller and Kochs, 2002), it appears that IFN activity in Huh-7 cells proceeds via a MxA-independent pathway since expression of MxA does not inhibit HCV replication and expression of a dominant-negative MxA does not interfere with the antiviral effects of IFN (Frese et al., 2001). Other studies have suggested that IFN-α blocks HCV replication through translational control involving PKR (Wang et al., 2003) and ISG56 (Sumpter Jr. et al., 2004). Thus, the underlying mechanisms of antiviral activity of type I IFNs as well as IFN-γ, IFN-λ, and IL-1 in the replicon system remain unresolved.

Ribavirin significantly improves the rate of sustained viral clearance compared to monotherapy with IFN-α (McHutchison et al., 1998; Di Bisceglie and Hoofnagle, 2002). Similarly, ribavirin and IFN-α in combination elicit strong synergistic inhibitory effects on subgenomic HCV replication (Tanabe et al., 2004; Kanda et al., 2004). The underlying therapeutic mechanism of ribavirin is unknown, but may involve induction of lethal mutagenesis, inhibition of RdRp activity, depletion of intracellular nucleotide pools via inhibition of the host enzyme inosine monophosphate dehydrogenase (IMPDH), or stimulation of the cellular immune response. In Huh-7 cells, ribavirin exhibits antiviral activity by acting as an RNA mutagen inducing error-prone HCV replication (Lanford et al., 2003; Zhou et al., 2003; Tanabe et al., 2004; Kanda et al., 2004). On the other hand, exogenous guanosine can suppress the mutagenic effect of ribavirin and potent IMPDH inhibitors enhance the antiviral capacity of ribavirin, suggesting that ribavirin can inhibit HCV replication by depleting GTP pools (Zhou et al., 2003). The HCV replicon system is being utilized to generate ribavirin-resistant variants. Interestingly, two conserved mutations in the C-terminal region of NS5A independently confer a low level of ribavirin resistance, while ribavirin-resistant HCV replication could also be attributed to defects in ribavirin import rather than mutations in the

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replicon RNA (Pfeiffer and Kirkegaard, 2005). Thus, HCV replicons will continue to provide a valuable model system for elucidating the mechanisms of ribavirin action, synergism with IFN, and ribavirin resistance in cultured cells.

Subgenomic replicons encode all the known cis-acting RNA sequences and viral enzymes (Table 1) required for replication that are now prime targets for antiviral drug design. Thus, the replicon system provides an excellent screening platform to identify compounds that effectively block enzymatic activities of HCV-encoded proteins and to evaluate the inhibitory effect of nucleic-acid based approaches including antisense oligonucleotides, ribozymes, and small interfering RNAs (siRNAs). Small-molecule inhibitors of the HCV NS3/4A serine protease that are effective in the nanomolar range have been identified (Lamarre et al., 2003; Pause et al., 2003) and nucleoside analogues and non-nucleoside small molecules have been explored as RdRp inhibitors (Carroll et al., 2003; Tomei et al., 2004; Beaulieu et al., 2004; Ludmerer et al., 2005; Tomassini et al., 2005). A peptidomimetic inhibitor of the protease, BILN 2061, provided the first proof-of-principle for preclinical evaluation of new antiviral drugs using the replicon system. BILN 2061 was very active at inhibiting genotype 1 subgenomic replication (IC50 3-4 nmol/L; Lamarre et al., 2003) and in a phase 1 clinical trial, BILN 2061 administered orally to HCV genotype 1-infected patients led to a 100-1000-fold drop in circulating virus within two days of treatment (Lamarre et al., 2003; Hinrichsen et al., 2004). In contrast, only half of the patients persistently infected with HCV genotype 2 or 3 who received BILN 2061 for 48 hours responded with a reduction in viral RNA greater than 1 log10 (Reiser et al., 2005), underscoring the need to develop replicon-based screening assays for the remaining HCV genotypes (genotypes 3, 4, 5 and 6). Another major factor limiting the efficacy of therapies to combat HCV infection will be the ability of HCV to develop resistance to specific antiviral drugs, but the HCV replicon system will allow potential drug-resistant variants to be rapidly identified and characterized. For example, drug-resistant substitutions in the NS3 protease domain emerge when replicon-containing cells are cultured in the presence of active inhibitors of the HCV protease (Trozzi et al., 2003; Lin et al., 2004; Lu et al., 2004) and a single mutation within the NS5B polymerase conferred resistance to a nucleoside analog, although replicons carrying this mutation were attenuated (Migliaccio et al., 2003). Replicon-containing cells are also being used to test the efficacy of RNA interference against HCV RNAs. Synthetic or stably expressed siRNAs and small hairpin RNAs targeting various regions of the HCV non-structural coding sequence (NS3, NS4B, and NS5B) as well as the 5' NTR efficiently suppress HCV replication, albeit with different efficiencies (Randall et al., 2003; Yokota et al., 2003; Seo et al., 2003; Wilson et al., 2003; Kapadia et al., 2004; Kronke et al., 2004; Takigawa et al., 2004). Interestingly, siRNAs are more effective at reducing HCV RNA levels than high doses (100 IU/ml) of IFN-α (Kapadia et al., 2004). Furthermore, replicating RNA can be cleared from >98% of siRNA-transfected

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cells (Randall et al., 2003), and siRNAs, introduced into Huh-7 cells prior to HCV replicons, effectively prevent the establishment of HCV replication (Wilson et al., 2003). Thus, these studies support the principle of siRNA-based HCV antiviral therapy; however, the challenges that have hindered nucleic acid therapies in the past still need to be resolved. As we have already begun to witness, the HCV replicon system will be fundamental for determining the antiviral potency of HCV inhibitors, optimizing drug regimens, monitoring for drug-resistance, and assessing the efficacy of nucleic-acid based antiviral strategies.

CONCLUDING REMARKSThe development of subgenomic replicons capable of autonomous replication in the human hepatoma cell line Huh-7 marked an important turning point for HCV research. The identification of cell culture-adaptive mutations and highly permissive Huh-7 sublines has enabled the development of transient replication assays as well as replication-competent monocistronic subgenomes, replicons that encode reporter genes, and full-length HCV genomes. Replicons derived from isolates belonging to genotype 1a, 1b, and 2a are now available and the cell tropism for HCV genotypes 1b and 2a has been expanded to other hepatoma cell lines, non-liver-derived cells, and murine hepatocytes. For the first time, HCV replication can be studied at the molecular level in cell culture. Many investigators have capitalized on this system to investigate important questions related to HCV biology that have plagued the field since the molecular cloning of the HCV genome 16 years ago. While significant advances have been made, there are many questions that remain which undoubtedly will be answered by future research. For instance, what are the mechanisms underlying cell culture adaptation of genotype 1 isolates? Why does the JFH-1 genotype 2a sequence not require adaptive mutations to replicate in cell culture? What are the factors that facilitate more efficient HCV replication in Huh-7 cells than in the other cell lines tested? Why do adaptive mutations prevent virus particle assembly? The recent discovery that full-length RNAs encoding the unmodified non-structural proteins from genotype 2a JFH-1 produce infectious virus particles overcomes one of the remaining barriers and now allows the complete viral life cycle to be studied in cell culture. Finally, subgenomic RNAs are already proving valuable for the development and evaluation of antiviral drugs as well as screening for the emergence of drug resistance. The replicon system should accelerate the development of effective drugs to cure individuals chronically infected with HCV.

ACKNOWLEDGMENTSWe are grateful to Dan Ader, John Majors, Sondra Schlesinger, and Milton Schlesinger for critical reading of the manuscript. Supported in part by the Ellison Medical Foundation New Scholars in Global Infectious Disease Research Program to K.J.B. (ID-NS-0119-03). E.A.N. is supported by the Monticello College Foundation

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Olin Fellowship for Women. We apologize to colleagues for the omission of other key literature citations due to space limitations.

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Chapter 12

Animal Models for HCV Study

Linda B. Couto and Alexander A. Kolykhalov

ABSTRACTThe study of HCV biology is complicated by the paucity of relevant animal models. The ideal model for studying HCV would be one that adequately represents most aspects of human HCV infection and disease, is affordable, easily available, and reproducible. Currently, the only widely recognized animal model of HCV infection is the chimpanzee, which does not meet all of these desirable attributes. Recently, other models have been used to dissect various aspects of HCV biology and to evaluate novel therapeutics. Each has a unique set of advantages and limitations. Transgenic mouse models have elucidated the pathophysiology of specific viral proteins, but they are limited by their inability to support HCV replication. Xenograft models provide an environment for human hepatocyte engraftment in mice and subsequent infection with HCV. These models are technically challenging, but once optimized they promise to be extremely useful both for the study of HCV biology and for drug development. Alternatively, the GBV-B virus, which efficiently replicates in tamarins and marmosets, represents a surrogate model for the study of HCV. Chimeras between GBV-B and HCV have been created and will be useful in the development of HCV-targeting drugs.

CHIMPANZEE MODELUse of the chimpanzee model to address questions regarding HCV biology is well justified by both the importance of HCV as a major healthcare problem and by the fact that the questions cannot be addressed otherwise. The chimpanzee is the closest genetic relative to human, which explains why many features of hepatitis C disease are so common between humans and chimps. Both humans and chimpanzees have detectable HCV RNA within a few days of infection. Maximum viral titers usually reach 105-107 RNA genome copies per mL of blood. The rise in viremia is usually followed by an increase in serum liver enzymes, which peak between 2 and 12 weeks. The majority of infected chimpanzees have necroinflammatory changes in liver biopsies; typically the disease is somewhat milder than that observed in humans. Antibodies to HCV antigens usually appear around week 8 or after. This acute phase of infection is followed by transition toward chronic viral persistence. It was initially reported that chimpanzees have lower rates of chronicity compared to humans, ~40% and ~70-85%, respectively. Bassett et al. (1998) reported that a cross-

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sectional study in the chimp colony at the Southwest Foundation for Biomedical Research, San Antonio, TX, revealed that out of 46 animals infected with different strains of HCV, only 18 (39%) were viremic based on reverse transcription-PCR analysis. More recent data on infection of naïve animals with HCV suggest that about 60% of all chimps became persistently infected; this rate of persistence is similar for HCV of different genotypes (Bigger et al., 2004; Nam et al., 2004). Chimpanzees with acute resolving infection usually clear virus in plasma during weeks 12 to 24.

As the only animal model for the study of HCV, the chimpanzee was used to provide early characteristics of HCV. Even before HCV had been identified, the chimpanzee was involved in the study of Non-A, Non-B hepatitis (NANBH) virus transmission, in establishment and duration of disease, and in the chronic nature of NANBH infection (Alter et al., 1978; Tabor et al., 1978). The first characteristics of the infectious agent, such as its size (Watanabe et al., 1987), and its inactivation with lipid solvents (i.e. the enveloped nature of the agent) (Bradley et al., 1983; Feinstone et al., 1983) were determined using the chimpanzees. Finally, the HCV genome was isolated and cloned for the first time from chimpanzee plasma with a high infectivity titer of Non-A, Non-B agent (Bradley et al., 1991; Choo et al., 1989).

The major advantages of the chimpanzee model stem from the ability to monitor and analyze the development of the disease from its initiation. Most clinical data on HCV infection in humans is derived from patients who have been infected for a period of time, often decades. Due to the asymptomatic nature of hepatitis C disease, the acute phase of the infection is often not noticeable, and thus, very little data exist regarding the events immediately following infection. In human studies, usually only samples of easily accessible tissues, such as blood, are available. Only a few liver biopsies per year can be performed in infected patients, which preclude efficient analysis of events in the primary tissue of HCV replication. On the contrary, liver biopsy samples from the chimpanzees can be obtained before the exposure and at planned intervals post-inoculation. These well controlled samples allowed the analysis of events starting immediately after HCV infection, such as changes in gene regulation and cellular immune responses to viral antigens. Furthermore, the possibility to rechallenge animals that cleared a previous infection, allowed memory immune responses to be analyzed (Bassett et al., 2001; Bigger et al., 2004; Farci et al., 1992; Ilan et al., 2002b; Prince et al., 1992; Weiner et al., 2001), as well as an analysis of HCV vaccine development (Forns et al., 2000; Houghton, 2000). Finally, the chimpanzee model was instrumental in the establishment of HCV infectious molecular clones (Kolykhalov et al., 1997; Yanagi et al., 1997). The virus recovered from an in vitro synthesized RNA resulted in the development in chimpanzees of classical signs of hepatitis disease, such as viremia, elevated serum

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levels of hepatic enzymes, histologic changes in the liver, and the development of HCV specific antibodies, thus formally proving that HCV is the causative agent of the disease.

REVERSE GENETICS/FUNCTIONAL ANALYSIS OF HCVThe ability to test the infectivity of molecular clones in chimpanzees allowed for the first time a reverse genetics analysis. This established the critical importance of all genome coded enzymatic activities, as well as some cis-acting elements in the HCV genome, for virus replication (Kolykhalov et al., 2000; Bukh et al., 1999). Though the HCV replicon tissue culture model is extremely useful for genetic analysis, it is restricted by the fact that replication of HCV RNA is not dependent on structural proteins (see Chapter 11). Thus, the chimpanzee model was required to demonstrate that the hypervariable region 1 (HVR1) of the envelope protein E2 is not critical for virus replication in vivo, and can be removed altogether (Forns et al., 2000). This result was somewhat unexpected since the HVR1 was considered among the primary regions of HCV to interact with the host immune system (Farci et al., 1996; Kato et al., 1993). In another experiment, it was demonstrated that p7 is absolutely essential for infectivity of HCV, and that the amino- and/or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function (Sakai et al., 2003).

MONOCLONAL INFECTIONSMany experiments using patient-derived virus were complicated by the fact that HCV exists as a set of quasispecies. Replication of the viral genome depends on the genome-encoded RNA dependent RNA polymerase, which lacks proofreading activity (see Chapter 10). As a consequence, viral replication results in the accumulation of numerous genetic variants, called quasispecies. The quasispecies nature of an inocula was thought to explain the initial evolution of the virus in vivo, as well as the escape of the virus from the immune response (Hijikata et al., 1995; Kojima et al., 1994; Okamoto et al., 1992). Recovery of virus from in vitro synthesized infectious RNA allowed the creation of "monoclonal" virus pools, derived from a single cDNA molecule. Chimpanzee serum, collected during the first weeks following intrahepatic inoculation of infectious RNA, exhibited no genetic variability (Major et al., 1999), therefore, providing a unique starting material for the study of viral evolution and of virus-host interactions. Infection of chimpanzees with such virus simplifies studies of HCV, since the interpretation of results is not complicated by the quasispecies nature of the inocula. Thus, Major at al. (2004) published detailed results of the analysis of ten chimps all inoculated with the same monoclonal virus, representing the dominant variant in the most studied patient isolate H77. Six out of ten infected animals became chronically infected, which implied that the presence of quasispecies in the inocula is not a requirement for establishing chronicity. The acute phase of infection was similar in all animals, whether they resolved the infection or became chronically infected. The


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maximal viral titers were 0.5-1 log higher in animals with chronic infection, usually between 106 and 107 RNA copies/ml. The viral load increased quickly during the first 1-2 weeks after infection (mean doubling time = 0.5 days) until reaching 103 to 105 copies/ml. This was followed by a significant delay in virus accumulation (mean doubling time = 7.5 days) over the next several weeks, during which titers increased by only 2 to 3 log10. Viral titers began to decrease in all animals as alanine amino transferase (ALT) responses increased, with peak RNA titers preceding ALT peaks by 2 to 3 weeks. ALT elevations in the serum are believed to be markers of hepatocyte death and could be due to killing of infected or bystander liver cells by the host immune response. Both the height and the time of the ALT peaks were similar between the groups. Following the ALT peak the viral titers decreased, and all infections resulted in 1 of 2 outcomes: persistence, with virus titers reaching a steady state at approximately 104 to 105 RNA copies/mL; or clearance, with titers continuing to decrease below levels of quantitation (<200 copies/mL in the study). After the decrease in viral titers, ALT levels returned to baseline in all animals despite significant levels of virus in those animals with persistent infection. The animals that became persistently infected were followed for 82-216 weeks after infection. Very low levels of the virus were observed in some animals after the clearance from time to time up to 1 year. This was attributed to the use of a highly sensitive RT-PCR method for virus detection (40 RNA copies/mL) and to very long follow-up of the cleared animals.

An HCV-specific antibody response in the Major at al. (2004) study was mounted during weeks 7-14 (usually on weeks 9-10) in the chronic group and during weeks 6-9 (majority on weeks 8-9) in the animals that resolved the infection. On the contrary, antibodies to the HVR1 were detectable only in the chronic group (in 5 out of 6 animals), but not in the resolved group. In general, anti-HVR1 antibodies correlate with anti-E1/E2 antibodies (Bartosch et al., 2003; Major et al., 1999). In an overlapping study by Logvinov et al. (2004) no neutralizing antibody (nAb) responses were detected in three animals that cleared the virus, whereas strain-specific nAbs were detected in six of the seven chronically infected animals after approximately 50 weeks of infection. These data suggest that nAbs do not play a role in the control of virus infection. This data correlates between human and chimpanzee (Prince et al., 1999).

CELLULAR IMMUNE RESPONSEThe role of the cellular immune response was also addressed in the chimpanzee model. The early events in a human infection are difficult to analyze, since liver tissue samples are not available during the acute phase of infection, nor are control liver samples taken from before the infection. Analysis of T-cell responses showed that animals who terminated the infection mounted a strong cytotoxic T lymphocyte (CTL) response (Cooper et al., 1999; Nascimbeni et al., 2003), and that CD8+ CTLs

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are better correlated with protection against HCV infection than antibodies. The appearance of these cells in the liver several weeks after infection was temporary associated with increases in liver enzymes in the plasma and with a temporary decrease in viral load in plasma . Thimme at al. (2002) showed that initial HCV spread outpaces the T cell response by demonstrating that HCV rapidly induces, but is not controlled by IFN-alpha and IFN-beta. Viral clearance follows the appearance and accumulation of HCV-specific IFN-γ-producing T cells in the liver. The importance of memory CD8+ T cells in the control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset in a chimpanzee (who had recovered from two previous infections) just before a third infection with the same dose and strain of HCV. Virus replication was significantly prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections, and it was not terminated until HCV-specific CD8+ T cells recovered in the liver (Shoukry et al., 2003). This was in sharp contrast to the second infection, when the effector function was not delayed, and the viremia was terminated within 14 days, i.e. 28 days earlier than during the third infection. Control of this second infection was kinetically linked to the rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in the blood. Though memory CD4+ T cells were intact in the CD8+ T cell-depleted animals, they did not facilitate rapid clearance of the virus. It does not, of course, rule out a critical supporting role for the helper T cells in protective immunity. This was addressed in other two chimpanzees that had cleared the infection. These animals were treated with an anti-CD4 monoclonal antibody before reinfection with HCV (Grakoui et al., 2003). The treatment resulted in significant reduction of circulating CD4+ T cells for over one year, that, in turn, resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ T cell responses. Incomplete control of HCV replication by memory CD8+ T cells in the absence of adequate CD4+ T cell help was associated with emergence of viral escape mutations in class I major histocompatibility complex (MHC)-restricted epitopes and in the failure to resolve HCV infection.

Most studies of T-cell mediated immunity to HCV suggest that this response is essential for resolution of infection. However some animals with high numbers of intrahepatic CD4+ and CD8+ T cells did not clear the infection completely. In these animals, a decrease of viral titers during the acute phase was followed by viral persistence. One explanation of this fact was provided by Erickson et al.(Erickson et al., 2001), who showed that HCV in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the viral population for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes


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were intact in animals that resolved hepatitis C spontaneously. Other explanations for the inefficiency of the HCV-specific CD8+ T cells include the possibility that at least a portion of the cells is anergic or arrested at an early stage of differentiation (Gruener et al., 2001; Ulsenheimer et al., 2003; Wedemeyer et al., 2002), that infected hepatocytes are resistant to immune recognition, and/or that HCV-specific CD8+ T repressor cells are present that produce anti-inflammatory cytokines, such as IL-10 (Accapezzato et al., 2004).

Prediction of the outcome of an HCV infection is complicated by the fact that despite the well documented correlation between the spontaneous resolution of infection and the presence of a vigorous cellular response, this correlation is not absolute. In the study by Thompson et al. (2003), it was shown that neither the chimpanzees that remained chronically infected, nor the animals that resolved the infection mounted a significant cellular response. Only weak and transient T helper responses were detected during the acute phase in all animals. This study suggests that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.

LIVER GENE EXPRESSIONChanges in liver gene expression in response to HCV infection were measured in acute phase samples of chimpanzees who either became chronically infected, temporarily controlled the infection, or cleared the infection (Bigger et al., 2001; Ilan et al., 2002b). In another study, chronic phase samples were compared for changes in gene expression in the livers of 10 chimpanzees (Bigger et al., 2004). Hundreds of genes were shown to be up or down regulated in response to HCV infection. Some changes in the expression profile are expected, such as changes due to the response to viral double-stranded (ds) RNA, which includes type 1 interferons (IFNs) and the IFN response genes. Changes due to the innate and adaptive immune response to the infection are also predicted, including activation and infiltration of NK cells, macrophages, and lymphocytes. In addition, changes due to the hepatocyte response to the cytokines expressed by the immune cells are expected. To determine the set of genes whose expression most likely reflects the initial host response to HCV in the liver, Su et al. (2002) attempted to identify genes with expression patterns that strongly correlated with the amount of HCV RNA in the serum of all of the chimps over the entire time course profiled, and found 27 such transcripts. Many of these genes were known to be stimulated by IFN-α, including STAT1, 2'-5' oligoadenylate synthase (OAS), and Mx1, which are well known to exert antiviral activity through inhibition of translation, activation and repression of transcriptional activity, and mRNA degradation (de Veer et al., 2001). Similarly, Bigger et al. (2001) found that during the first phase of infection the most notable changes in gene expression occurred in numerous IFN response genes that increased dramatically, some as early as day 2 post-infection. Moreover, Su et al. (2002) showed that although

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HCV-infected cells successfully induce the transcription of many antiviral IFN-α-stimulated genes, this response has little or no effect on viral titer or outcome. Analysis of gene expression in 10 chronically infected chimpanzees confirmed that many IFN-stimulated genes were transcriptionally elevated, suggesting an ongoing response to IFN and/or dsRNA (Bigger et al., 2004). On the contrary, transient and sustained viral clearance was uniquely associated with the induction of IFN-γ-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response (Su et al., 2002). IFN-γ-induced genes are known to be expressed as a result of the homing and activation of immune cells to the liver (Boehm et al., 1997). Surprisingly, induction of IFN-γ itself was not detected in Su et al. (2002) microarray analysis, probably due to its low expression levels. The increases in IFN-γ mRNA levels were detected by RT-PCR in all tested animals in the Major at al. (2004) study, and these increases coincided with ALT elevations and decreases in viral titers in the plasma. In the cohort of animals infected with the same monoclonal virus, the induction of IFN-γ was observed both in those animals with self-limiting infections and in chronically infected animals. Additionally, the level of induction did not correlate with spontaneous clearance of the virus; IFN- γ induction was at least as high in the group of animals that progressed to chronic infections as for those that eventually cleared HCV. Also, the levels of IFN- γ mRNA remained elevated into the post-acute phase to a similar degree in all animals. Out of four genes reported in this study for which data were collected, two genes had profiles of the expression that correlated with the outcome; induction of the CD3e and MIP-1a were observed only in animals that cleared the virus. The initial peaks of CD3 also coincided with the control of virus replication.

A single genotype 3-infected animal was available for analysis in study performed by Bigger et al. (2004), and this animal exhibited increased expression of a number of genes potentially involved in steatosis compared to the levels of expression in animals with genotype 1 infections.

Unlike human patients, for whom the timing of infection, route of infection, the source and nature of the virus are often not known, the chimpanzee provides a well controlled clinically relevant model for the study of HCV. However, it is extremely limited in its availability, as well as by its expense. As a consequence, many results have been generated in experiments with low numbers of animals. In order to generate more statistically credible data, alternative animal models that are readily available and less expensive are needed.

TRANSGENIC MOUSE MODELSA number of transgenic mouse models have been developed to examine the potential pathogenic effects of the HCV core protein and/or the envelope glycoproteins on hepatocytes. Conflicting results have been reported, and therefore it has been


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hard to make any conclusive statements regarding the pathogenicity of the HCV structural proteins. In one study, the core protein of HCV (genotype 1a) was shown to be produced in mouse liver at levels similar to that seen in chronically infected HCV individuals, and pathogenesis was not observed over the course of 18 months (Pasquinelli et al., 1997). However, in another similar transgenic line expressing the core protein from HCV (genotype 1b), vacuolations in the liver were observed which led to steatosis in animals at 3-12 months of age (Moriya et al., 1997). Steatosis is the abnormal accumulation of fat within hepatocytes, and it appears to be a factor affecting chronic hepatitis C progression in humans (Rubbia-Brandt et al., 2004). In follow-up studies, Moriya et al. (1998) reported the formation of gross hepatic nodules in animals at 16 months of age, and the development of hepatocellular carcinoma (HCC) in some animals, suggesting that the core protein plays an important role in HCC. In addition, an age dependent increase in oxidative stress within the liver, as measured by an increase in lipid peroxidation and a decrease in glutathione levels, was observed in the transgenic mice that developed HCC (Moriya et al., 2001). As endogenous oxidants are an important class of naturally occurring carcinogens that act by producing genetic alterations, this may contribute to the etiology of HCC. In a transgenic line that was created to express not only core, but also E1 and E2 of HCV (genotype 1b), hepatitis was observed at 10-15 months, but no neoplastic nodules or carcinomas were observed during 4 years of cumulative observation in animals that ranged in age from 4-20 months.(Honda et al., 1999). Another transgenic line expressing these same three transgenes showed no evidence of liver pathology during the six months these animals were evaluated (Kawamura et al., 1997). In addition, no histological abnormalities associated with the expression of the envelope proteins alone were observed in transgenic mice up to 18 months of age (Koike et al., 1995; Pasquinelli et al., 1997). The conflicting results regarding the pathology associated with the HCV structural proteins could be due to differences in the mouse strains used to generate the transgenic animals. Alternatively, the discrepancies could be attributed to the different promoters that were used to express the transgenes, which resulted in different levels of protein.

One of the drawbacks of using transgenic models to study the potential pathogenesis of HCV proteins is the fact that the animals are tolerant to the transgenic protein, and thus, the role of the immune response to HCV proteins cannot be evaluated. To overcome this limitation, Wakita et al. (1998) constructed a transgenic model that allows conditional expression of the core, E1,E2, and NS2 proteins of HCV (genotype 1b) using the Cre/loxP recombination system. An adenoviral vector expressing Cre DNA recombinase was used to induce the expression of the HCV proteins. Upon infection of these mice with the adenoviral vector, acute hepatitis was observed and a humoral response to core protein was detected, indicating that the transgenic animals were immunocompetent for HCV proteins. To determine the role of the cellular immune response in the development of hepatitis, CD4+ and CD8+

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T cells were depleted by administration of anti-CD4 and anti-CD8 monoclonal antibodies. In the absence of T cells, no histopathological differences were observed between Ad-infected and uninfected transgenic mice, suggesting that HCV structural proteins are not directly cytopathic to hepatocytes, but rather a cellular immune response to these proteins is responsible for the hepatitis observed. However, a caveat to this interpretation is that adenoviral vectors alone cause hepatitis in a T cell-dependent manner, so it is not clear if the hepatitis observed was caused by the HCV structural proteins or the adenovirus (Yang et al., 1996). In a follow-up study (Wakita et al., 2000), these authors reported that injection of the Ad-Cre vector increased the CD8+ lymphocyte infiltration in the livers of transgenic mice more than that in non-transgenic mice, and ALT levels were higher in the former. In addition, CTLs isolated from the livers of transgenic mice were HCV specific, suggesting that HCV structural proteins are indirectly responsible for liver injury, and that the host immune response plays a role in the pathogenesis of HCV. The Cre/loxP system is a useful model to evaluate host/viral protein interactions, but it could be improved by expressing the Cre protein from a non-inflammatory viral vector, such as an adeno-associated viral vector.

Although still controversial, HCV infection has been reported to be associated with several extrahepatic manifestations, including hypertrophic and dilated cardiomyopathy. Using transgenic mice expressing the HCV core protein, Omura et al. (2005) recently demonstrated the development of histological changes consistent with cardiomyopathy after 12 months of age. However, the pathogenicity of these cardiac complications is not well understood.

XENOGRAFT MODELSTwo xenograft models for studying HCV have been developed and are now being used to evaluate HCV biology and anti-HCV therapies. Both models rely on transplantation of human hepatocytes into mice and subsequent repopulation of the mouse liver. One model utilizes Alb-uPA transgenic mice, which carry a tandem array of four murine urokinase-type plasminogen activator genes under the control of a liver-specific albumin promoter (Heckel et al., 1990). Over-expression of the transgene is cytotoxic to hepatocytes and results in a hypofibrinogenemic state and fatal neonatal bleeding. Spontaneous inactivation of the transgene occurs in a portion of the cells, giving them a growth advantage over transgene-containing cells, and the uPA-negative cells eventually repopulate up to 90% the liver (Sandgren et al., 1991). Mercer et al. (2001) combined the properties of these mice with those of immunodeficient SCID mice to develop a model system that allows human hepatocyte engraftment in mouse liver, and used this as a small animal model to study HCV infection. In this model transgenic uPA mice are transplanted with a million human hepatocytes, and engraftment occurs over the course of 4-6 weeks. Following engraftment the animals are inoculated with HCV-infected serum from


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human donors. Evidence for replication of HCV in these animals was confirmed by demonstrating viral titers of 1x104-1x106 copies/ml for periods up to 35 weeks. In addition, the authors claimed to detect negative-strand RNA, supporting the contention that bona fide HCV viral replication occurs in the animals. Viral replication was further validated by serially passaging the virus through three generations of mice, during which viral RNA levels increased 37,500 times. Considering dilution of the virus during passaging, this could not be attributed to the original human inoculum. These results clearly demonstrate that both replication of the HCV genome and production of fully infectious viral particles is possible in this animal model.

Recently, this chimeric mouse/human model was used to evaluate novel anti-HCV therapies. In one report (Hsu et al., 2003), the mice were used in a gene therapy approach to treat HCV by delivering a modified form of the BH3-interacting death agonist (BID). BID is a member of the Bcl-2 family of pro-apoptotic proteins and is crucial for death receptor-mediated apoptosis (Esposti, 2002). It is activated upon cleavage by caspase 8, and induces an increase in the permeability of the outer mitochondrial membrane, leading to release of apoptogenic proteins, such as cytochrome c. In this study, the endogenous cleavage site of BID was engineered to contain a specific cleavage site recognized by the NS3/NS4A protease of HCV. An adenoviral vector encoding the modified BID was delivered to the livers of SCID/Alb-uPA mice that had been previously infected with HCV. Animals were evaluated for serum HCV RNA titers, as well as, clinical and liver pathology. HCV-infected animals had initial HCV titers that ranged from 1x104-5x107 genome equivalents/ml. The animals with lower titers were able to completely clear the infection following Ad-BID vector administration, while a 2-3 log decrease in HCV viral titers was observed in animals with higher initial titers. Histological examination of the livers of animals inoculated with both HCV and Ad-BID showed extensive cell death, and a TUNEL assay confirmed apoptosis in their livers.

A company called KMT Hepatech (Edmonton, Alberta) has now been founded on the basis of this chimeric human/mouse technology, that provides "KMT mice" to collaborators interested in evaluating potential HCV therapies. Although not yet published in a peer-reviewed journal, a scientific poster on the company's website details the use of this model to evaluate two anti-HCV therapeutics that have been used successfully to treat HCV in humans (P-187 10th HCV Meeting Dec2-6,2003 Kyoto, Japan; KMT Hepatech website). The two drugs tested were IFN-α-2B and the novel protease inhibitor, BILN2061, which has been shown in human clinical trials to reduce viral RNA levels by 2-3 logs (Lamarre et al., 2003). Homozygous SCID/Alb-uPA mice transplanted with human hepatocytes were treated with either IFN-α-2B or BILN2061, and statistically significant reductions in HCV viral loads were observed with both agents. Thus, two therapies that have been shown

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to be effective against HCV in humans, and one novel gene therapy, are able to reduce viral titers in HCV-infected "KMT mice", thereby validating the model for evaluation of novel anti-HCV therapies.

Another mouse model that has been developed to study HCV replication is a modification of the "Trimera mouse". Trimera mice are created by total body irradiation of normal mice, followed by reconstitution with bone marrow from SCID mice. These animals are subsequently engrafted with human hematopoietic cells or solid tissues, such as liver. Since the resulting animal is comprised of three genetically distinct sources of tissue, the name Trimera mouse was coined. These mice were originally created to study the development of human B and T cells, but they are now also used to study HCV. When human liver fragments are transplanted, engraftment rates of up to 85% have been reported one month post-implantation (Ilan et al., 2002a). When healthy human liver tissue fragments were infected ex vivo with HCV positive serum, viremia was detected in 50% of transplanted animals. Mean viral loads of up to 1x105 copies /ml peaked on day 18, and subsequently declined by day 25. Direct transplantation of infected human livers also resulted in viremia in mice for approximately one month. The decline in viremia is the result of fibrosis and necrosis of the human liver tissue, which limits the evaluation of potential anti-HCV therapeutics. In addition to the presence of viral RNA in the serum of mice, HCV RNA was also detected in the implanted human liver tissue. Whereas positive-strand RNA was observed on day 0, negative-strand RNA was not observed until day 9, suggesting that HCV replication occurs. The trimeric model has been shown to support replication of HCV 1a, 1b, 2a, and 3a.

Having established that bona fide HCV replication occurs in the Trimera mouse model, it was used to evaluate the efficacy of two novel anti-HCV agents. A small molecule HCV IRES inhibitor and an anti-HCV monoclonal antibody showed modest dose-dependent reductions in the viral load of HCV during the treatment period, which returned to pretreatment levels following cessation of drug treatment (Ilan et al., 2002a). Viability of the hepatocytes was assessed by measuring the levels of human serum albumin (HSA) mRNA in grafts from control and treatment groups. Similar levels of HSA mRNA were observed, demonstrating that the reduced viral load was not due to a hepatotoxic effect of the drug. In these studies the maximal initial viral load was approximately 7x104 copies/ml. This level of viremia will need to improve before potent anti-viral agents, that have the ability to knock down HCV titers by several logs can be tested. Another issue with the model is that viremia persists for only one month, limiting the timeframe available to test the efficacy of drugs. At present, protocols to increase this window using antifibrotic agents are being attempted.


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Maeda et al. (2004) recently proposed an alternative to the Trimera model, which involves engrafting human liver tissue into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Although very good engraftment (approximately 90%) was observed in these mice, inoculation of these animals with either HCV-infected human serum or culture media containing an infectious HCV molecular clone resulted in HCV viral titers that were near the limit of detection of the PCR-based assay. HCV sequences were detected in the engrafted liver tissue by in situ PCR, but at present this does not represent a robust animal model for studying HCV replication or for evaluating anti-HCV therapies.

All xenograft models that have been described rely on RT-PCR for quantification of HCV RNA. To reliably detect changes in titer, the viral load should be at least 10,000 copies/ml, in order to see efficacy in the range of a one log decrease in titer. The main advantage of these models is that they represent a potentially less expensive in vivo model for studying HCV relative to the chimpanzee. Another advantage of the xenograft models is that HCV infection and replication occurs in human hepatocytes as opposed to chimpanzee or other non-human primate hepatocytes. Compared to the tissue culture replicon systems, adaptive mutations in the HCV genome do not seem to be required for replication in the xenograft models. However, none of these models has yet to achieve widespread utility due to the technical difficulty in breeding and creating the mice, the limited availability of human hepatocytes, variable human cell engraftment and inconsistent viral titers. If these difficulties can be overcome, these models will greatly aid the study of HCV biology and pathogenesis, as well as facilitate the development of new therapies for HCV. In order to expand the application of these mouse models for studying the role of the immune system in the pathogenesis of HCV, it will be necessary to reconstitute the mice with components of the human immune system.

SMALL NON-HUMAN PRIMATE MODELSThe chimpamzee is the only truly validated animal model for studying HCV, but because they are an endangered species, expensive to work with, and the subject of ethical debates, other non-human primates were evaluated for infection by HCV. Early studies concluded that NANBH virus could infect marmosets (Feinstone et al., 1981) and tamarins (Karayiannis et al., 1983), both of which are New World monkeys. However, these studies were undertaken before the NANBH virus group had been subdivided into identifiable agents and before specific diagnostic tests for HCV were available. Once these were obtained, it was concluded that chimpanzees, but no other non-human primates, were susceptible to HCV infection (Garson et al., 1997).

Despite this, the pursuit of a small-animal model to study HCV has not abated, and the GBV-B virus, which efficiently replicates in tamarins and marmosets, is an

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example of a surrogate model of HCV that has been gaining credibility. Originally identified as a "GB hepatitis agent", it was transmitted to tamarins from the blood sample of a surgeon (whose initials are GB) that was suffering from acute hepatitis (Deinhardt et al., 1967). All infected tamarins developed an acute hepatitis, and subsequently, the GB hepatitis agent was identified as containing two distinct RNA viruses: GBV-A and GBV-B (Simons et al., 1995). It was later shown that GBV-A does not replicate in the tamarin liver, whereas GBV-B causes hepatitis. Sequence analysis of the GBV-A and GBV-B genomes suggested that they belong to the Flaviviridae family. The GBV-B virus contains a positive-sense, single-stranded RNA genome of 9399 nucleotides, and it was shown to be most closely related to HCV. Additional experimental infection of tamarins using either the original serum sample, or serially passaged infected tamarin serum, confirmed that GBV-B causes acute hepatitis (Beames et al., 2000; Bright et al., 2004; Bukh et al., 1999). In three different species of tamarins, viremia with peak viral titers in excess of 1x108 genomic equivalents/ml was observed between 2 and 14 weeks post-injection, which subsequently cleared by 16 weeks post-injection. Viremia was accompanied by an increase in liver enzymes and inflammation in the liver. Because tamarins, like chimpanzees, are in limited supply, another New World monkey, the common marmoset, has been assessed for susceptibility to GBV-B infection. Marmosets are easier to manage as breeding colonies and are currently bred for biomedical research in a number of facilities. Several reports have confirmed the susceptibility of marmosets to GBV-B infection (Bright et al., 2004; Jacob et al., 2004; Lanford et al., 2003). Viral titers of over 1x108 genome equivalents/ml peaked around 6 week post-injection and viral clearance was complete by week 16. Increases in some liver enzymes were observed, which correlated with inflammation in the liver, as the result of infiltration of CD8+ lymphocytes. This GBV-B marmoset model was recently tested as a small-animal model for HCV by evaluating several anti-HCV therapeutics. A small-molecule inhibitor of the HCV NS3 protease was also shown to inhibit GBV-B replication in vivo (Bright et al., 2004). This inhibitor reduced GBV-B viral replication by more than three logs. This was the first time an anti-HCV therapeutic was demonstrated to be effective in an animal model other than the chimpanzee, and provides validation of the GBV-B/marmoset model as a surrogate for studying HCV. Not only is this model valuable for evaluating therapies directed against the NS3 protease, it may soon be shown to be useful for testing therapies targeting other regions of the HCV genome.

One of the major differences between the course of infection of GBV-B in New World monkeys to that of HCV in humans is the tendency for the former to result in an acute infection and the propensity of the latter to lead to chronic hepatitis. However, in one recent study viremia was observed for >2 yrs following infection of one tamarin with GBV-B (Martin et al., 2003). In this case, the animal was infected by direct intrahepatic inoculation of synthetic RNA. It is not clear if this


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was related to the outcome, but in any case, it enhances the value of the GBV-B tamarin or marmoset model.

The cloning and sequencing of GBV-B revealed some differences and similarities between GBV-B and HCV (Muerhoff et al., 1995) and allowed the construction of chimeric viruses to be made between the two. The genomic organization and structure of GBV-B and HCV are similar; each containing a single long ORF flanked by 5' and 3' nontranslated regions (NTR). The 5' portion of the ORF was predicted to encode structural proteins, while the 3' portion of the ORF encodes nonstructural proteins (Muerhoff et al., 1995). Even though the homology of the predicted polyproteins between GBV-B and HCV is low (25-30%), the hydropathy plots of the polyproteins are very similar. The early work that led to the realization of chimeric viruses was the demonstration that the GBV-B and HCV NS3 protease share substrate specificity (Scarselli et al., 1997). This guided the construction of a functional chimeric GBV-B/HCV protease that consisted of an N-terminal HCV protease domain and a C terminal GBV-B RNA helicase domain. The chimeric NS3 retained protease activity capable of processing both GBV-B and HCV substrates, and retained helicase activity similar to that of the native GBV-B NS3 (Butkiewicz et al., 2000). The ability to construct a chimeric NS3 polypeptide with HCV protease and GBV-B helicase activities suggested that it may be possible to create viable chimeric GBV-B/HCV viruses that could be used to test protease inhibitors in the tamarin and/or marmoset model. For this to be realized, the development of an infectious clone of GBV-B was required. Although the GBV-B genome was initially cloned and sequenced in 1995, RNA transcribed from this clone was not infectious. It was not until 1999, when it was shown that the 3'NTR extends an additional 259 nucleotides, that an infectious clone was generated (Bukh et al., 1999). This set the stage for the construction and evaluation of chimeric viruses. This area is currently in its infancy and chimeric viruses may eventually serve as models for testing HCV protease, helicase, or polymerase inhibitors, as well as, therapeutic agents that target the viral RNA.

A recent publication (Rijnbrand et al., 2005) describes a functional chimeric virus derived from GBV-B, in which a functionally important HCV regulatory sequence was substituted for the analogous sequence in the 5' NTR of GBV-B genome. Domain III of the GBV-B NTR, which binds directly to the 40S ribosome subunit, was replaced with the corresponding domain from HCV. Inoculation of tamarins with RNA transcripts derived from this chimeric clone led to recovery of viable virus, which resulted in acute hepatitis. This result demonstrates that domain III of HCV can substitute for the similar domain in GBV-B and can support both translation and viral replication in vivo. However, the kinetics of viremia were noticeably different, and the unusual infection profile was shown to be due to the accumulation of compensatory mutations that arose to support efficient viral

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replication. This is an exciting new model for the evaluation of HCV replication and for use in drug screening, as this chimeric virus will allow the investigation of potential anti-HCV therapies targeted to domain III of the HCV IRES.

Similar chimeras may soon be available in the 3'NTR region of GBV-B, following the functional analysis of mutant 3'NTR sequences. The GBV-B 3'NTR consists of a short sequence of 27 nucleotides, followed by a poly(U) tract of 23 nucleotides, and a 3' terminal sequence that consists of 309 nucleotides. By deleting specific regions of the 3'NTR and testing the mutants by intrahepatic transfection of tamarins with the transcribed RNAs, functionally important areas of the 3'NTR were identified. Deletion of both the poly (U) tract and the short proximal sequence killed the virus; however, deletion of just one of these elements resulted in viable viruses (Nam et al., 2004). The authors also showed that insertion of a long heterologous sequence in the proximal sequence resulted in the recovery of a virus that had deleted the majority of the insert, but retained a short fragment of the heterologous sequence. This suggests that it should be possible to insert short DNA sequences into the GBV-B 3'NTR.

Studies using chimeric replicating viruses in the marmoset model, while providing a new modality for testing anti-HCV therapies, will certainly have limitations. These viruses may not completely mimic the HCV viral life cycle and the insertion of HCV elements into the GBV-B sequence may not accurately reflect the natural accessibility of these elements in the HCV genome. However, it represents a new small-animal model alternative to the chimpanzee and will be useful in evaluating at least some potential drug candidates.

SUMMARY AND CONCLUSIONSThe chimpanzee remains the best animal model for studying the biology of HCV, as it is the only animal that is susceptible to HCV infection and replication, and because the liver disease observed in chimpanzees mimics the pathology seen in humans. Nevertheless, this model is difficult to use due to its expense, inaccessibility, and ethical considerations, and thus, efforts to develop other smaller animal models have continued. A major advantage of a small-animal model is that large numbers of animals can be employed, thus, providing statistically significant data. A few mouse models have been in the development now for about one decade. Transgenic mice expressing HCV structural proteins, for example, can be used to detect potential pathophysiological features of specific viral proteins. However, these animals cannot be used to probe questions about the viral life cycle. In addition, in all but one transgenic model, the mice are tolerant to the HCV proteins preventing the immune response against these proteins from being evaluated. In one model, conditional expression of HCV proteins is possible. Since the host immune response to HCV is believed to play an important role in disease, this latter model may be very


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useful. Several mouse/human xenograft models are also being used to study HCV biology and to evaluate potential anti-HCV therapeutics. These models, although very promising, still suffer from a lack of reproducibility and require skilled and experienced individuals to create them. Another drawback, is that the animals are immunocompromised, and thus virus/host interactions cannot be assessed fully. Finally, the use of GBV-B/HCV chimeric viruses that infect and replicate in New World monkeys offer the advantages of direct virus infection of a small animal, without the complication and irreproducibility of hepatocyte engraftment. Although these viruses may not perfectly recapitulate HCV biology, drugs that target specific regions of these chimeric viruses can be evaluated using this model. All of the animal models for studying HCV have their limitations, but careful selection of a model will allow investigators to ask specific questions regarding HCV infection, replication, pathogenesis, and/or drug sensitivity, and important information can be gleaned.

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HCV and Host Defense

Chapter 13

HCV Regulation of Host Defense

D. Spencer Carney and Michael Gale Jr.

ABSTRACTMammalian cells respond to virus challenge by initiating a "host response" characterized by interferon α/β (IFN) production and a cellular antiviral state. The host response is our first line of immune defense against viral pathogens and it imposes several barriers that hepatitis C virus (HCV) must overcome to replicate and persist. HCV evades the host response through a complex combination of virus-host interactions that disrupt intracellular signaling pathways and attenuate the antiviral actions of IFN. Regulation of the host response breaks a link between innate and adaptive immunity and provides a foundation for HCV replication and spread.

INTRODUCTIONExposure to HCV typically leads to persistent infection associated with a chronic disease course. The ability of HCV to mediate persistent, life-long infection in its human host is linked to the evasive nature of the virus to thwart the host immune system and to resist the antiviral actions of IFN-based therapy. Molecular studies of HCV-host interactions have revealed several levels of immune regulation and evasion directed by HCV protein products. This chapter provides an overview of the virus-host interface of these regulatory processes and their impact on HCV replication and persistence.

INNATE INTRACELLULAR IMMUNE DEFENSESIn response to virus infection, signaling pathways within mammalian cells direct a variety of intracellular events that generate an antiviral state directly within the infected cell. This antiviral response, termed the 'host response" to virus infection, represents our first line of immune defense against virus infection. If this response is successful, exposure to the virus will render a self-limiting, abortive infection. It is the hepatic host response that imposes initial immune defenses against HCV infection (Gale, Jr., 2003). The host response is triggered when the infected cell recognizes a molecular signature within the invading virus. This signature, known as a pathogen-associated molecular pattern (PAMP), is typically a physical characteristic of the virus that is recognized and engaged by specific PAMP receptor proteins within the host cell (O'Neill, 2004). The PAMP/PAMP receptor interaction initiates signaling cascades that induce the expression of antiviral effector genes

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(Sen, 2001). For RNA viruses, protein and nucleic acid products of infection comprise an array of PAMP signatures that can engage specifi c PAMP receptors, including Toll-like receptors (TLRs) and nucleic acid binding proteins (Fig. 1; Iwasaki and Medzhitov, 2004; Cook et al., 2004). The HCV RNA contains specifi c PAMP signatures, including poly-uridine motifs and stem-loop double-stranded RNA (dsRNA) structures within its single-stranded RNA genome (Tuplin et al., 2002; Simmonds et al., 2004). HCV RNA is suffi cient to induce the host response in cultured hepatocyte-derived cell lines (McCormick et al., 2004; Sumpter et al., 2005). The product of retinoic acid inducible gene I (RIG-I), which has been defi ned as a dsRNA PAMP receptor, is critical for host response signaling induced by HCV RNA (Yoneyama et al., 2004; Sumpter et al., 2005). In hepatocytes, the independent signaling pathways of RIG-I and Toll-like receptor 3 (TLR3) direct the host response to virus infection (Li et al., 2005a).

PAMP/PAMP receptor engagement signals the activation of a variety of transcription factors in the infected hepatocyte. PAMP-driven transcription factor activation results in the immediate expression of host response genes (Fig. 2; Malmgaard, 2004). Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) are triggered in response to virus infection, and each are activated upon cellular recognition of the HCV PAMP (Au et al., 1995; Lin et al., 1999; Fredericksen et al., 2001; Richmond, 2002; Prabhu et al., 2004). Their activation proceeds through PAMP-responsive signaling pathways of the cell that promote their nuclear translocation and transactivation functions. Other IRF family members, including IRF-5 and IRF-7, are essential components of the host response to virus infection (Barnes et al., 2001; Kawai et al., 2004), though the specifi c role of each in HCV infection has not been characterized. Virus-induced signaling events that activate ATF-2 and further direct chromatin remodeling contribute to the building of a virus-triggered enhancesome on the IFN-β promoter. The IFN-β enhanceosome includes

Fig. 1. Triggering the host response to HCV infection. HCV triggers the host response through the process pathogen-associated molecular pattern (PAMP)/PAMP receptor engagement. Certain RNA motifs within the HCV genome have PAMP attributes that activate the host response. RIG-I (Sumpter et al., 2005) and possibly TLR3 confer HCV PAMP recognition. Signaling through these and possibly other pathways leads to the activation of IRF-3, IFN production and ISG expression. Autocrine and paracrine signaling by IFN can potentiate viral PAMP signaling and serves to amplify the host response through a feedback loop involving IRF-7 and PAMP signaling molecules. IFNs also enhance immune cell maturation and effector function, resulting in a host response aimed at controlling virus infection.

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IRF-3 and NF-κB, which produce a transcriptional response resulting in IFN-β expression and secretion from the infected cell (Sen, 2001). NF-κB activation and function is central to the chemokine and proinfl ammatory cytokine response to virus infection, which functions side by side with IFN to modulate the ensuing adaptive immune response (Tai et al., 2000). Secreted levels of IFN-β drive autocrine and paracrine signaling processes by binding the IFN-α/β receptors of the infected cell

Fig. 2. Signaling the host response to HCV infection. 1. Viral PAMP (HCV RNA) binding to RIG-I , TLR3 or other PAMP receptors results in the phosphorylation and activation of IRF-3 by the TBK1 or IKKε protein kinases. The dimer of phospho-IRF-3 translocates to the cell nucleus, interacts with its transcription partners (Yoneyama et al., 1998), and binds to the cognate DNA positive regulatory domain (PRD) in the promoter region of IRF-3 target genes, including IFN-β. 2. IRF-3 activation drives IFN-β production. 3. IFN-β binding to the IFN α/β receptor signals the activation of the associated Tyk2 and Jak1 protein kinases and the phosphorylation/ assembly of a STAT1/STAT2 heterodimer and trimeric ISGF3 (Sen, 2001). 4. ISGs are induced by ISGF3. ISGs are the genetic effectors of the host response, and IRF-7 is itself and ISG and a critical transcription factor whose actions are turned on through viral PAMP signaling pathways that overlap with the pathways of IRF-3 activation. IRF-7 phosphorylation, dimerization and heterodimerization with IRF-3 directs its binding to the virus-responsive element (VRE) in the promoter region of IFN-α genes, resulting in IFN-α subtypes expression (Au et al., 2001). This increases the abundance of RIG-I and viral PAMP signaling components to amplify host response. The therapeutic administration of IFN-α provides antiviral action against HCV by signaling ISG expression through the IFN α/β receptor and the Jak-STAT pathway. The NS3/4A protease prevents IFN-β production by disrupting RIG-I signaling and cleaving TRIF to ablate TLR3 signaling (Foy et al., 2005; Li et al., 2005b).

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and local surrounding tissue, respectively. This results in activation of the Jak-STAT pathway. Here, receptor-associated Jak and Tyk1 protein kinases phosphorylate signal transducer and activator of transcription (STAT) proteins on critical serine and tyrosine residues to confer STAT activation, STAT association with IRF-9, and nuclear localization of the resulting ISGF3 transcription factor complex. ISGF3 is the central transcription factor that promotes high level expression of the interferon stimulated genes (ISGs) by binding to the IFN-stimulated response element (ISRE) within the ISG promoter/enhancer region. IFN binding to the IFN α/β receptor and signaling of the Jak-STAT pathway drives a second wave of transcriptional activity initiated by virus infection and denoted by the expression of ISGs.

ISG PRODUCTS HAVE ANTIVIRAL FUNCTIONSThe human genome encodes hundreds of ISGs (Der et al., 1998). The ISG products direct regulatory functions that control virus infection. ISGs have been shown to interrupt HCV replication through processes that include suppression of viral protein synthesis and synthesis inhibition of the viral negative strand replicative intermediate (Guo et al., 2001; Shimazaki et al., 2002; Wang et al., 2002; Prabhu et al., 2004). The main feature resulting from the secretion of IFN from infected cells into the local tissue is the paracrine induction of a tissue-wide host response that blocks cell to cell spread of the virus. Many components of the host response pathways are themselves ISGs, and though expressed basally at a low level that facilitates surveillance and response to virus infection, their abundance will increase in response to IFN signaling. In the liver, paracrine signaling of IFN serves to enhance overall responsiveness of cellular signaling pathways to potentiate the host response to infection in a tissue-wide manner. IFN signaling thus provides an amplification loop to further promote the host response against HCV (Foy et al., 2005). Recent studies have shown that the amplification loop is dependent upon the transcriptional activity of IRF-7 (Honda et al., 2005). IRF-7 is an ISG and its expression in the liver is IFN-dependent (Smith et al., 2003; Honda et al., 2005). IRF-7 promotes the expression of the various IFN-α subtypes, wherein IFN-α production and secretion mediate further amplification of the host response and prolonged IFN production (Honda et al., 2005). IFN-based therapy for HCV infection exploits these actions of IFN-α to limit HCV replication and spread (Fig. 2) (McHutchison and Patel, 2002). IFN-α also signals the maturation of immune effector cells, antigen presenting cells and dendritic cells, and it potentiates the production of other proinflammatory cytokines by resident hepatic cells to indirectly modulate cell-mediated defenses and adaptive immunity (Biron, 1999). Viral triggering and control of the host response may therefore define cellular permissiveness for HCV RNA replication and influence the outcome of infection.

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CROSS-TALK BETWEEN INNATE AND ADAPTIVE IMMUNE DEFENSES.IFN-α is a potent immunomodulator that influences the onset of the cellular immune response and adaptive immunity to HCV infection. IFN-α modulates natural killer (NK) cell activity toward lysis of infected target cells by promoting NK cell activation and proliferation, and supporting their survival through the induction of IL-15 production (Biron, 1999; Loza and Perussia, 2004). Activated NK cells produce IFN-γ (Shoukry et al., 2004). In the HCV-infected liver, NK cell homing and local secretion of IFN-γ may serve to limit HCV replication directly. Indeed, IFN-γ has been shown to mediate direct antiviral effects against HCV RNA replication in vitro (Frese et al., 2002), and its production by immune effector cells in the liver associates with viral clearance in the chimpanzee model of HCV infection (Thimme et al., 2001; Su et al., 2002). IFN-α also influences the maturation of dendritic cells and modulates their presentation of viral antigens (Colonna et al., 2004; Barth et al., 2005). Antigen presentation by dendritic cells under the influence of IFN-α contributes to the differentiation of CD4 T cells toward the Th1 phenotype and importantly, a Th1-predominate response is associated with clearance of HCV infection (Shoukry et al., 2004). IFN-α production during HCV infection may therefore indirectly contribute toward directing the maturation of CD4 T cells to the Th1 phenotype. Dendritic cells are also involved in the cross priming and IFN-γ production of CD8 T cells. In this context the co-stimulatory signals presented at the time of antigen cross presentation to CD8 T cells can determine whether the cells are cross primed for a cytotoxic response or cross-tolerized for anergy (Cooper et al., 1999; Shoukry et al., 2004). TLR3 plays an essential role in cross priming by dendritic cells, in which it signals the production of IFN-α triggered by viral PAMPs within products of phagocytosis (Schulz et al., 2005). IFN-α produced by the dendritic cell in this manner induces the expression of co-stimulatory molecules and cytokines to promote the cross priming and activation of CD8 cells, thus linking host response signaling processes to induction of the adaptive immune response. The link between innate, IFN-induced antiviral pathways and the adaptive immune response is not entirely understood and further studies are required to define the linkage of these processes with the outcome of HCV infection.

IFN-γ ACTIVATION OF THE ANTIVIRAL RESPONSEIFN-γ may serve to complete the signaling loop between infected hepatocytes and immune effector cells. The IFN-γ receptor is expressed by hepatocytes and in most other tissues. In the liver, IFN-γ produced by infiltrating NK cells and activated T cells, where it can bind its receptor in paracrine fashion to induce a hepatic IFN-γ response. IFN-γ receptor binding leads to phosphorylation of STAT1 through the receptor-bound Jak1 and Jak2 protein kinases (Stark et al., 1998). The phosphorylated STAT then forms a homodimer that translocates to the cell nucleus and acts on the gamma-activated sequence elements of target genes to promote their

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transcription (Der et al., 1998). In hepatocytes the genes under the control of the GAS element have significant overlap with those expressed in response to IFN α/β and under control of the ISRE (Cheney et al., 2002). GAS elements are also found within genes whose products are involved in antigen processing and presentation, immune effector action and apoptosis, thus deriving a variety of antiviral actions. A role for GAS element genes in host defense against HCV is supported by studies of the chimpanzee model for HCV infection, where the high expression of IFN-γ responsive genes in the liver has associated with the resolution of acute infection in the chimpanzee model (Su et al., 2002). Together with IFN-α, the actions of IFN-γ provide addition levels of host defense cross-talk that serves to limit HCV infection.

HEPATIC DEFENSES ARE TRIGGERED BY HCVFunctional genomic analyses from cohorts of patients infected with HCV have shown that infection associates with a hepatic gene expression profile marked by ISGs whose expression levels vary widely among patients (Smith et al., 2003). These observations indicate that HCV can induce and regulate the hepatic host response to infection. Gene profiling studies of HCV-infected chimpanzees have demonstrated that acute resolving HCV infection is associated with a host response characterized by high level hepatic ISG expression (Bigger et al., 2001). In similar studies, "outcome predictor" gene sets were identified by the overall hepatic expression level of certain ISGs and virus-responsive genes. This gene set was accurately defined as those virus-responsive genes whose high expression associated with low viral load and effective viral clearance but whose low expression correlated with progression to chronic HCV infection (Su et al., 2002). Like many virus-responsive genes and ISGs, the various products of the "outcome predictor" gene set are known to interact with components of T cell immunity, again demonstrating the complex cross-talk that goes on during the host response to infection and between parameters of innate and adaptive immunity. These observations demonstrate that the hepatic host response is triggered during HCV infection but differentially regulated in association with disease course, and that HCV mediates persistence through strategies to regulate and/or evade the antiviral actions of this response.

TRIGGERING THE HOST RESPONSE TO HCV INFECTIONThe processes by which HCV initiates and controls the host response have been addressed in cell culture models of HCV RNA replication and viral protein expression. Genome-length or specific subgenomic fragments of HCV RNA are sufficient to trigger IFN-β expression and production when introduced into cultured human hepatoma cells (Fredericksen et al., 2001; McCormick et al., 2004), and this effect has been attributed in part to the 5' and 3' nontranslated region (NTR) of the HCV genome. These regions encode a series of highly conserved stem-loop/dsRNA structures, and the 3' NTR includes a variable length poly u region (Tuplin

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et al., 2002). In cultured cells the HCV NTRs present PAMP structures that serve as potent agonists of the host response. This suggests that during infection these RNA motifs are recognized and engaged by PAMP receptor(s) that trigger the host response (Sumpter et al., 2005).

The nature of at least one HCV RNA PAMP receptor was revealed through studies of the Huh7-derived (human hepatoma) cell line, termed Huh7.5. This cell line does not mount a host response to virus infection or transfected HCV RNA and it is highly permissive for HCV RNA replication (Blight et al., 2002; Sumpter et al., 2005). Complementation studies identified RIG-I as an HCV RNA PAMP receptor that binds HCV dsRNA motifs and signals the activation of IRF-3 and NF-κB. This was shown to induce IFN-β expression and onset of the host response (Sumpter et al., 2005). RIG-I is an RNA helicase and a member of the DEx/D box RNA helicase family. It contains amino-terminal regions of homology to the caspase activation and recruitment domain (CARD) (Yoneyama et al., 2004). RIG-I signaling is mediated by the CARD homology motifs while the helicase domain imparts PAMP recognition, RNA-binding and regulation of signaling (Yoneyama et al., 2004). Signaling by RIG-I directs a host response that suppresses HCV RNA replication (Sumpter et al., 2005; Foy et al., 2005). The permissiveness of Huh7.5 cells for HCV RNA replication has been attributed to a point mutation in the RIG-I CARD motifs that ablated downstream IRF-3 phosphorylation and NF-κB activation (Sumpter et al., 2005). Virus-induced signaling by RIG-I and the resulting host response may therefore influence the outcome of HCV infection. TLR3 is a dsRNA PAMP receptor that signals a host response after its engages the PAMP ligand (Fig. 2) (Alexopoulou et al., 2001). TLR3 signals the activation of IRF-3 and NF-κB through MyD88-independent processes that require the Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) protein (Yamamoto et al., 2003). Genetic and biochemical studies have now defined prominent roles for the RIG-I and TLR3 pathways in signaling the host response to virus infection in hepatocytes (described below), though the role of each in natural HCV infection remains to be evaluated.

Protein products of infection may also stimulate the host response to HCV. Expression of the HCV NS5A protein induces cellular stress signaling pathways to activate STAT3 (Gong et al., 2001). Similar to the IFN α/β receptor signaling, STAT3 promotes gene expression through processes that involve the Jak-STAT pathway (Sarcar et al., 2004). This results in a gene expression profile that includes ISGs and proinflammatory cytokines that may influence the overall level of HCV RNA replication (Zhu et al., 2003). Moreover, the HCV core protein has been shown to activate the catalytic activity of protein kinase R (PKR), an ISG effector component of the host response to virus infection (Delhem et al., 2001). PKR is a dsRNA binding protein. Its activation is induced by binding to dsRNA PAMPs,

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resulting in eukaryotic initiation factor 2 phosphorylation and in inhibition of local protein synthesis. Active PKR also signals the DNA binding activity of NF-κB and induces IRF-1 transcription-effector action (Williams, 1999). Activation of PKR by the HCV core protein is most likely attributed to the 5' NTR viral RNA binding property of the core protein (Tanaka et al., 2000), which would provide the PKR activator dsRNA PAMP required for kinase activation. Cell interaction with virus particles may also trigger signaling events that induce IFN production. HCV pseudo particle binding to dendritic cells has been shown to mediate particle uptake and dendritic cell activation (Barth et al., 2005). Since dendritic cell subsets constitute a major source of IFN production during viral infection, HCV modulation of dendritic cell function may influence HCV infection by modulation local or systemic IFN levels (Colonna et al., 2004).

REGULATION AND EVASION OF THE HOST RESPONSE BY HCVHCV utilizes a variety of strategies to regulate and evade the host response. Studies of the HCV/host interface have revealed PAMP-responsive signaling pathways that impart IRF-3 activation, IFN α/β receptor signaling, and ISG effector action as major sites of control and evasion of the host response (Katze et al., 2002). The HCV NS3/4A protease has been identified as an antagonist of virus-induced IRF-3 activation and IFN-β expression. NS3/4A mediates a block to IRF-3 activation triggered either by endogenous replicating HCV replicon RNA or by exogenous virus infection of cells harboring a replicating HCV genomic RNA (Foy et al., 2003). The IRF-3 blockade was been attributed to the NS3/4A protein complex, which mediates a block to virus-induced IRF-3 phosphorylation, resulting in retention of IRF-3 in an inactive, cytoplasmic-bound state. NS3 is a bifunctional enzyme, and it encodes serine protease within its amino-terminal domain and a RNA helicase within its carboxyl-terminal domain (Reed and Rice, 1998). Importantly, the NS3/4A complex constitutes the essential viral protease, and it releases the nonstructural proteins from the HCV polyprotein during virus replication (De Francesco and Steinkuhler, 2000). The helicase activity of NS3 is not required for the control of IRF-3 activation but the NS3/4A protease activity is required. This suggests that HCV blocks IRF-3 activation through NS3/4A proteolysis of essential host cell proteins that confer PAMP signaling (Foy et al., 2003). NS3/4A regulation of RIG-I signaling has been identified as a causal link of the IRF-3 phosphorylation blockade directed by NS3/4A. These studies showed that NS3/4A blocks the host RIG-I pathway through the protease-dependent disruption of CARD-homology domain signaling that is normally induced upon RIG-I binding to the HCV RNA PAMP ligand. The NS3/4A block to RIG-I signaling additionally ablates virus activation of NF-κB (Foy et al., 2005). This dual regulation of IRF-3 and NF-κB indicates that NS3/4A must target and cleave common factor(s) involved in IRF-3 and NF-κB activation but the nature of such factors have not been defined.

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TLR3 signaling is also targeted and regulated by NS3/4A. In this case NS3/4A protease activity has been shown to cleave the TRIF adaptor protein between amino acids C372 and S373 (Li et al., 2005b). This cleavage site is homologous with the HCV NS5A/B polyprotein cleavage site. Structure/function studies show that NS3/4A recognizes TRIF through binding of a proline-rich region adjacent to the site of cleavage (Ferreon et al., 2005). TRIF is essential for signaling by TLR3 but it does not play a role in RIG-I pathway, nor does TRIF cleavage by NS3/4A provide a mechanism of RIG-I pathway regulation, which must occur through cleavage of yet addition cellular targets (Foy et al., 2005; Li et al., 2005a). NS3/4A cleavage of TRIF prevents TLR3 signaling, thus blocking IRF-3 and NF-κB activation and preventing IFN production (Fig. 2) (Li et al., 2005b). The targeting of the RIG-I and TLR3 pathways by NS3/4A allows HCV to evade two major arms of IFN production and host defense. This has been further validated through pharmacologic studies that used a peptidomimetic active site NS3 protease inhibitor to evaluate the requirement for protease activity in the regulation of host response signaling by NS3/4A. Treatment of cells that expressed wild type, functional NS3/4A showed that the protease inhibitor effectively removed the blockade to RIG-I and TLR3 signaling imposed by HCV, thereby restoring virus-induced IRF-3 phosphorylation/activation and the activation NF-κB (Foy et al., 2003; Foy et al., 2005). Protease inhibitor treatment of cells also protected TRIF from cleavage by NS3/4A (Li et al., 2005b).

What are the implications resulting from HCV disruption of RIG-I or TLR3 signaling? First, viral control of RIG-I and TLR3 serves to attenuate major pathways of IFN production by infected cells and tissues. Second, many of the components of these pathways are IFN-responsive and though expressed at low levels, their expression is increased after exposure of cells and tissues to IFN α/β. The IFN-responsiveness of these factors provides an amplification loop to enhance the strength and length of the host response. The signaling blockade imposed by NS3/4A breaks this IFN amplification loop (Foy et al., 2005) and may limit the level and diversity of ISG expression induced in response to IFN therapy. Third, HCV attenuation of the host response and IFN production is expected to cause alterations in antigen presentation by the affected hepatic tissue, potentially leading to inefficient activation of cytolytic T cells and an inability of the adaptive immune response to clear HCV-infected hepatocytes by disrupting the cross talk between the host response and the adaptive immune response. This may provide a causal link between high level intrahepatic ISG expression and a vigorous T cell response to diverse viral epitopes, both of which are associated with viral clearance and inversely correlate with chronic infection (Su et al., 2002; Shoukry et al., 2004). Fourth, IRF-3 has been ascribed proapoptotic and tumor suppressor functions (Heylbroeck et al., 2000; Duguay et al., 2002). A prolonged block to IRF-3 activation might disrupt these actions and cause a tumorigenic phenotype within infected cells. This

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could provide a link between chronic HCV and hepatocellular carcinoma (Liang and Heller, 2004). Finally, the blockade of NF-κB function may interfere with a variety of chemokines and cytokine genes whose expression is dependent on NF-κB and serve to drive the inflammatory response to virus infection (Zhu and Liu, 2003; Foy et al., 2005). HCV regulation of NF-κB may therefore contribute to the systemic immune defects associated with HCV infection.

HCV REGULATION OF IFN SIGNALINGThe overall low response rate of HCV to IFN therapy, particularly among patents with genotype 1 HCV infection (McHutchison et al., 2002), provides clinical evidence that HCV can effectively evade IFN actions in vivo. Many studies have focused on defining the molecular mechanisms by which HCV evades and resists IFN actions. These studies have shown that HCV protein expression associates with inhibition of STAT1 function, and can occur independently of STAT tyrosine phosphorylation (Heim et al., 1999). Analysis of transgenic mice that express HCV proteins in their hepatocytes showed that the regulation of STAT1 occurs below the level STAT tyrosine phosphorylation, resulting in a defective hepatic IFN response (Blindenbacher et al., 2003). STAT dysfunction might be attributed to protein phosphatase 2A (Fig. 3), which confers STAT1 hypomethylation and complex formation the protein inhibitor of activated STAT1 (PIAS). This was found to prevent STAT1 assembly into the ISGF3 complex and to attenuate ISG expression (Duong et al., 2004). The mechanism by which protein phosphatase 2A triggers these events is not known. Others have shown that expression of the HCV core protein associates with increased levels of suppressor of cytokine signaling (SOCS)-3 (Bode et al., 2003). SOCS3 belongs to a family of SOCS proteins that are negative regulators and inhibitors of Jak-STAT signaling. SOCS proteins mediate a classical negative feedback loop on IFN α/β receptor signaling events; SOCS-1 and SOCS-3 confer reduced levels of ISG expression (Alexander, 2002). While induction of SOCS-3 by the HCV core protein might impart evasion from IFN actions, it should be noted that the overall role of SOCS-3 in HCV infection is not known and it is unclear if expression of SOCS-3 by core is due to the potential for the core protein to stimulate IFN signaling events itself (Miller et al., 2004) or through induction of yet undefined signaling pathways that stimulate SOCS expression.

REGULATION OF ISG EXPRESSION OR FUNCTIONMolecular studies have linked HCV evasion of the host response and IFN therapy with various strategies directed by viral proteins to control ISG expression or function (Table 1). The HCV NS5A protein has been identified as an IFN antagonist. Several studies have shown that expression of NS5A alone can attenuate IFN-α actions and rescue the replication of IFN-sensitive viruses (Macdonald and Harris, 2004). Microarray analyses have shown that NS5A expression can confer a general attenuation of ISG expression, though the mechanism of this regulation was not

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defi ned (Geiss et al., 2003). NS5A induces interleukin (IL)-8 expression and secretion. This has implications for IFN therapy because IL-8 is a proinfl ammatory chemokine whose actions interfere with IFN (Fig. 3). The mechanism(s) of IL-8's anti-IFN action may include attenuation of IFN signaling and ISG expression, and/or direct inhibition of select ISGs (Khabar et al., 1997). Serum IL-8 levels are found elevated in patients with chronic HCV, and NS5A has been shown to stimulate IL-8 production through transactivation of the IL-8 promoter, possibly involving NF-κB and AP-1 transcription factor activation by other cytokines (Polyak et al., 2001a, Polyak et al., 2001b). IRFs may also drive IL8 production when activated during virus infection (Casola et al., 2000). This latter point is another example that serves to demonstrate the link between innate antiviral and pro-infl ammatory responses. In this case the link is exploited by NS5A as a means of evading the host response to HCV infection.

Fig. 3. HCV attenuation of IFN signaling. IFN α/β Receptor signaling by IFN from autocrine/paracrine and therapeutic sources is subject to feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. The HCV core protein can induce the expression of SOCS-3, which suppresses Jak-STAT signaling events (Alexander, 2002). Expression of the protein inhibitor of activated STAT (PIAS) is induced by HCV proteins, possibly mediated by protein phosphatase 2A (PP2A) signaling events and STAT demethylation (Duong et al., 2004). This blocks STAT1 function. Some patients with chronic HCV infection exhibit aberrantly high levels of serum IL-8 (Polyak et al., 2001b). The biological activity of IL-8 interferes with IFN signaling events (Khabar et al., 1997). HCV modulation of IFN signaling allows the virus to evade the antiviral actions of the host response and IFN therapy.

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Table 1. Processes of ISG regulation or control by HCV.Viral strategy Mechanism of

actionImplications Reference/Example

IL-8 induction. NS5A induces IL8 production through processes involving NF-κB and AP-1 transcription factor activation.

Attenuates ISG expression.

Polyak et al., 2001a

Induction of SOCS expression.

The HCV core protein can induce expression of SOCS1 and SOCS3.

Blocks Jak-STAT signaling action through the IFN α/β receptor.

Bode et al., 2003

PKR inhibition NS5A and E2 proteins bind PKR and inhibit its catalytic activity.

Disruption of PKR-dependent translational control and signaling actions.

Taylor et al., 1999; Noguchi et al., 2001; Gimenez-Barcons et al., 2005

IRF-1 regulation NS5A blocks ds RNA induced IRF-1 action through inhibition of PKR signaling.

Relieves IRF-1 suppression of HCV RNA replication.

Pflugheber et al., 2002; Kanazawa et al., 2004

Evasion of 2'-5' OAS/RNase L pathway

HCV genome sequence.

The HCV genome encodes a paucity of RNase L recognition sites, which allow protection from nucleolytic processing.

Han et al., 2002; Han et al., 2004

Disruption of STAT1 function.

HCV proteins HCV proteins induce PP2A expression and STAT1 hypomethylation to attenuate ISG expression.

Heim et al., 1999; Duong et al., 2004

Suppression of ISG56 expression.

HCV nonstructural proteins

In vitro: NS3/4A and nonstructural proteins disrupt virus signaling to the ISG56 promoter. Removes the ISG56 block to viral RNA translation.

Wang et al., 2002; Sumpter et al., 2004

Regulation of RIG-I signaling

NS3/4A protease blockade of signaling.

Blockade of RIG-I signaling breaks an IFN amplification loop that otherwise enhances ISG expression.

Foy et al., 2005

Regulation of TLR3 signaling

NS3/4A protease cleavage of TRIF

Disruption of a TLR3-pathway IFN amplification loop.

Li et al., 2005b

Activation of STAT3 The NS5A protein induces a stress response that activates STAT3.

STAT3-target genes, including some ISGs, are expressed.

Gong et al, 2001

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The NS5A and E2 proteins of HCV have both been identified as inhibitors of PKR (Gale, Jr. et al., 1998; Taylor et al., 1999; Noguchi et al., 2001). Inhibition of PKR may allow HCV to evade in part the translational-suppressive and signaling actions of PKR (Katze et al., 2002). This regulation is not universal and is subject to alteration through viral genetic variation, such that not all NS5A sequences have the ability to bind and inhibit PKR (Gimenez-Barcons et al., 2005). Thus, HCV evasion of PKR-independent processes of the host response must also contribute to HCV resistance to IFN. The product of the IFN-induced ISG56 gene (also known as IFIT1 or the 561 gene), p56, can suppress HCV RNA translation (Wang et al., 2002), and viral attenuation of ISG56 expression has been shown to associate with a level of resistance to HCV RNA replication from the antiviral actions of IFN in the HCV replicon cell culture model. ISG56 is both an ISG and an IRF-3-target gene (Grandvaux et al., 2002), and the IFN-resistant phenotype in this case associated with viral genetic adaptations that enhanced the NS3/4A blockade to IRF-3 signaling (Sumpter et al., 2004). This raises the possibility that HCV control of IRF-3 activation pathways may attenuate ISG expression by preventing the cross-talk of cellular pathways that converge on ISG promoter elements. Studies that have evaluated HCV interactions with the IFN-induced 2'-5' oligoadenylate synthetase (OAS)/RNase L antiviral pathway have shown that HCV proteins interact with this pathway (Taguchi et al., 2004). When activated, this pathway directs RNase L, an endoribonuclease, to cleave the HCV genome RNA into nonfunctional products (Han and Barton, 2002). RNase L cleaves HCV RNA only at certain UU and UA dinucleotide sites (Han et al., 2004), and genotype 1 HCV sequences have overall fewer RNase L cleavage sites than HCV genotypes 2 or 3 (Han et al., 2004). This could provide a genetic mechanism for how, in part, HCV 1a and 1b infections resist IFN therapy (Table 1). However, as described above and owing to the pleiotropic nature of IFN effects mediated by the hundreds of ISGs, it is likely that HCV evades IFN action through multiple strategies to redirect ISG functions.

VIRAL GENETICS IMPACT THE HOST RESPONSE TO HCV INFECTIONThe viral polymerase of HCV lacks proofreading function (Reed et al., 1998), and during persistent infection the error-prone virus replication generates a repertoire of highly related but genetically distinct viral variants or "quasispecies". This provides a remarkable adaptive potential to HCV and has been implicated in evasion and control of the host response and IFN therapy (Farci, 2001). A hostile host environment may drive the outgrowth of HCV "evasion variants" from a pre-existing quasispecies pool or through viral genetic adaptation. This idea is supported by in vivo studies that evaluated viral sequences from the E1 and E2 coding regions within patients with HCV infection. This work demonstrated viral genetic patterns that associated with infection outcome and in which the resolution of acute HCV infection consistently associated with an overall reduction in viral quasispecies

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complexity (Farci et al., 2000; Farci et al., 2002). In contrast, progression to chronic infection and resistance to IFN therapy associated with increased viral genetic complexity, suggesting that host immune pressure drives the outgrowth or selection of viral evasion variants able to persist and resist IFN action. Analysis of the HCV NS5A coding region has also identified specific domains that exhibit sequence variation in patients with differential outcomes to IFN therapy. Meta-analyses and long-term follow-up of these studies now provide support for NS5A sequence variation within a 40 aa "interferon sensitivity determining region" (ISDR) that associates with IFN therapy outcome (Enomoto et al., 1996, Pascu et al., 2004; Schinkel et al., 2004). The ISDR is within a genetically-flexible domain that is a major site of viral adaptations among HCV RNA replicons (Blight et al., 2000; Appel et al., 2005). Thus, ISDR variation may affect the HCV replication fitness and the host response to infection.

EXOGENOUS INDUCTION OF ANTIVIRAL HEPATIC DEFENSESVarious studies have described an absence or only a low level expression of IFN α/β within liver tissue from patients with chronic HCV infection (Mihm et al., 2004). This lack of high IFN α/β gene expression within the HCV-infected liver provides indirect evidence that HCV imposes a blockade to IRF-3 activation in vivo and may explain why some patients with chronic infection do not express significant levels of hepatic ISGs. However, it fails to explain why other patients exhibit broad and abundant ISG expression despite only low level IFN α/β expression in the infected liver (Smith et al., 2003). The fact that hepatic ISG expression has associated with the level and extent of liver pathology (Smith et al., 2003) suggests that ISGs might be induced indirectly as a result of cellular stress from fibrosis and/or cirrhosis. ISGs are also induced through STAT3 signaling; studies have shown that STAT3 is activated by NS5A, and that STAT3 activation occurs concomitantly with HCV RNA replication (Gong et al., 2001; Waris et al., 2005). Viral activation of STAT3, possibly mediated through stress-responsive signaling events, may contribute to ISG expression during HCV infection (Fig. 4). ISGs may also be induced through TLR engagement exogenously by extracellular products of damaged tissue or viral replication. It is also noted that stress-induced cytokines, including TNF-α and IL-1 can trigger IRF-1 expression and transactivation function, resulting in a level of IFN-β production (Fujita et al., 1986). This could contribute to hepatic ISG expression even when the RIG-I or TLR3 pathways are blocked by the HCV NS3/4A protease. Exogenous immune effector cells that infiltrate the liver may also contribute to hepatic ISG expression. In particular, IFN production by tissue macrophages and dendritic cells that have infiltrated the infected tissue could lead to a paracrine IFN response and ISG expression during the processes of antigen cross-presentation in vivo (Schulz et al., 2005). By this model hepatic ISG levels would vary with the composition and extent of immune cell infiltration, which has been observed (Smith et al., 2003). Secretion of IFN-γ by T cells and NK cells

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that have infi ltrated the infected liver also contributes to ISG expression but the pattern of expressed ISGs only partially overlaps with those induced by IFN α/β (Der et al., 1998).

THE HCV/HOST INTERFACE MODEL OF VIRAL EVASIONFig. 5 depicts a model of the virus/host interface and host response regulation that forms a foundation for persistent HCV infection. The transmission event of HCV infection presents unique pressures for the virus to adapt to the new host environment. HCV adaptation to a new host will involve fi ne tuning of viral strategies to control and evade the host response to infection. The transmission event results in an acute infection that involves viral regulation of the host response though RIG-I, TLR3 and other host defense signaling pathways within the infected cell (Sumpter et al., 2005; Li et al., 2005a). Highly fi t variants of HCV will mediate signaling interference at the virus/host interface. This involves the actions of the NS3/4A protease to block RIG-I and TLR3 signaling pathways. However, this regulation is infl uenced by viral genetic variation, and genetic distinctions among the many different quasispecies of HCV that are replicating in various cells at various times post-infection will result in differential levels of control and activation of this response. During acute infection the differential activation and control of the host response by viral genetic variants will lead to the production of IFN and ISG expression to mediate an antiviral state in the local hepatic tissue (Bigger et al., 2001). 15-25% of all exposures to HCV typically result in acute/resolving infection (McHutchison, 2004), indicating that a robust hepatic host response could provide protection against the replication and spread of HCV. The host response and the ensuing adaptive immune response present enormous pressures that will select for the outgrowth of viral quasispecies that can evade and successfully control

Fig. 4. Processes of hepatic IFN production from exogenous sources. Various processes can result in hepatic ISG expression despite a block to the host response imposed by the actions of HCV proteins. In most of the examples shown, IFN will be produced from non-infected (exogenous) cell sources and not from the chronic HCV-infected hepatocyte, which studies have shown do not express appreciable levels of IFN (Mihm et al., 2004). NS5A triggering of STAT3 signaling (Waris et al., 2005) would produce ISGs in the absence of IFN, and this affect would be limited to infected cells.

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the host response and immune defenses (Farci et al., 2000; Sumpter et al., 2004). HCV/host interactions at key sites of host defense signaling serve to suppress the host response, attenuate the actions of IFN therapy, and provide a solid foundation for persistent HCV infection. This model incorporates the important, variable and perhaps unpredictable aspect of viral adaptation and quasispecies selection within the evasion and control strategies by which HCV limits the host response to infection. Further studies to identify the viral genetic elements (including viral PAMPs and PAMP structure), signaling factors and ISG effectors that regulate the host response to infection will certainly increase our understanding of host defense and the HCV/host interface that controls infection outcome.

ACKNOWLEDGEMENTSThe Gale laboratory is supported by grants from the NIH, the Ellison Medical Foundation and the Burroughs Wellcome Fund. DS is supported by NIH training grant 5T32DK007745. M.G. is the Nancy C. and Jeffrey A. Marcus Scholar in Medical Research in Honor of Dr. Bill. S. Vowell.

Fig. 5. HCV/host interactions regulate infection outcome. The model is described in the text, and its shows a fl ow of events in which the virus/host interface and interactions within the host response will decide the fate of HCV infection. By this model, HCV disruption of the host response to infection provides a the frame work for viral persistence and chronic infection, may contribute resistance to IFN therapy.

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NOTE ADDED IN PROOFRecent studies have shown that NS3/4A cleaves the host Cardif/iPS-1 protein to block RIG-1 signaling during infection. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and Tschopp, J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172.

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Does HCV Cause Dysfunction in the Immune Cells?

Chapter 14

Regulation of Adaptive Immunity by HCV

Xiao-Song He

ABSTRACTHCV causes chronic infection in the majority of infected patients, which is associated with attenuated adaptive immunity against the virus. Accumulating data suggest that HCV may modulate the adaptive anti-HCV immunity of the host to facilitate the establishment of viral persistence. Potential mechanisms of this modulation include infection of dendritic cells by HCV, as well as binding of HCV envelope or core proteins to cell surface receptors, resulting in perturbation of the functions of different immune cell subsets. These mechanisms may operate predominantly in the liver, the primary site of infection by HCV, where the unique hepatic environment favors tolerance rather than immunity to foreign antigens. Elucidation of these mechanisms may lead to development of novel therapeutic strategies combining both antiviral drugs and immunotherapy agents.

INTRODUCTIONHepatitis C virus (HCV) is a major blood-borne virus that infects over 100 million people worldwide and 2.7 million in the United States. It is estimated that in less than 20% of HCV-infected individuals the virus is cleared spontaneously, while in the majority of patients the virus persists and causes chronic hepatitis that may lead to end-stage liver diseases requiring liver transplantation (Alter et al., 1999). The mechanisms underlying different outcomes of infection are not clear at this time. The host immune responses, including innate immunity and adaptive immunity, play a critical role in determining the outcome of viral infection, as well as in the nature and extent of liver cell injury during HCV infection (Rehermann and Chisari, 2000; He and Greenberg, 2002). Since the rate of persistence for HCV is much higher than other hepatitis viruses, for example, hepatitis B virus (HBV) that persists in only less than 10% of immunocompetent adults who are infected (Hollinger and Liang, 2001), HCV appears to be more successful than many other viruses in terms of evading the protective immunity of the host. However, little is known regarding the exact reasons for the failure of the host immune system in fighting HCV. In this article the current knowledge regarding the adaptive immunity to HCV will be reviewed first, followed by a discussion on the potential mechanisms HCV may employ to interfere with the normal functions of host immune system to achieve its persistence.

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ADAPTIVE IMMUNITY TO HCV: A FAILURE IN MOST PATIENTSAlthough HCV persists in the majority of infected individuals, a small fraction of patients can successfully clear the infecting virus. In a study on injection drug users, those who resolved previous HCV infection were 12 times less likely to be reinfected to develop persistent infection than people infected for the first time. In those who did become reinfected, the median peak HCV RNA levels were two logs lower than people infected for the first time to develop persistent infection. These findings suggest that a protective immunity does exist, which is capable of complete or partial control of HCV infection (Mehta et al., 2002).

While the role of neutralizing antibodies in the protective immunity against HCV has recently regained attention (Hsu et al., 2003; Logvinoff et al., 2004), most of the previous studies on the human adaptive immune responses against HCV focused on the T cell responses. Although the number of cases with self-limited HCV infection that have been carefully studied is relatively small, such studies usually reveal a vigorous HCV-specific T cell response, including CD4 T cell response (Gerlach et al., 1999; Takaki et al., 2000; Thimme et al., 2001; Rosen et al., 2002) and CD8 T cell response (Gruner et al., 2000; Lechner et al., 2000b; Takaki et al., 2000; Thimme et al., 2001; Lauer et al., 2004). These responses were detected early during the acute phase (Takaki et al., 2000; Thimme et al., 2001) and sustained for many years after the clearance of HCV (Takaki et al., 2000). They were usually broadly targeted at multiple epitopes restricted by different MHC molecules, without a dominant epitope (Cooper et al., 1999; Lauer et al., 2004). In contrast, patients with chronic HCV infection usually have weak or defected T cell responses against HCV, as indicated by low frequencies for the specific T cells (He et al., 1999; Lauer et al., 2004), short-lived responses (Lechner et al., 2000a; Ulsenheimer et al., 2003), narrowly targeted epitopes (Lauer et al., 2004), as well as defects in the effector functions of the specific T cells (Gruener et al., 2001; Wedemeyer et al., 2002). Taken together, these studies strongly suggest that the host T cell responses are a key factor in determining the outcome of HCV infection. Of note, during the acute phase of self-limited HCV infection, a brief period of dysfunction of HCV-specific CD8 T cells has also been documented (Lechner et al., 2000b; Thimme et al., 2001), suggesting that a transient down-modulation of the effector functions of specific CD8 T cells may be a host strategy to limit the tissue damage caused by the cytotoxic CD8 T cells at the early stage of infection when viral replication is at its peak rate.

Although it is still unclear why T cell responses fail to clear HCV in most cases, a comparison of T cell immunity against HCV and other viruses with different outcomes of infection has generated some intriguing results. Within the category of persistent viruses, the pattern of viral replication varies from latent infections that undergo periodic reactivation (e.g. Epstein-Barr virus, EBV), to ongoing low-level

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viral replication (e.g. human cytomegalovirus, CMV) controlled by the immune responses without causing disease, to persistent high-level viral replication subjected to immune control to variable extents at different stages of disease course, as in the case of HIV. MHC class I tetramers and cytokine flow cytometry assays have been used to characterize phenotypes of human CD8 T cell responses to persistent viral pathogens (He et al., 1999; Appay et al., 2000; Gamadia et al., 2001; Hislop et al., 2001; Catalina et al., 2002; Khan et al., 2002; Wedemeyer et al., 2002). A study comparing the phenotypes of peripheral CD8 T cells specific for HIV, CMV, EBV and HCV showed that the expression of CD8 T cell surface markers thought to be related to CD8 T cell differentiation, including CD27 and CD28, were heterogeneous between CD8 T cells specific for different viruses (Appay et al., 2002). The authors proposed the use of CD27 and CD28 expression to classify virus-specific CD8 T cells into early (CD27+CD28+), intermediate (CD27+CD28–) and late (CD27–CD28–) differentiated cells. Interestingly, CD8 T cells specific for each of the four viruses appeared to fall into different stages based upon this classification; that is, HCV-specific CD8 T cells had markers associated with the early differentiation phenotype, EBV-specific CD8 T cells were classified as early-intermediate, CD8 T cells recognizing HIV antigens were intermediate, and only CMV-specific CD8 T cells had the proposed late phenotype. Of note, memory CD8 T cells specific for influenza A virus (fluA), which causes transient infections and is cleared by the immune system, have phenotypes consistent with those of early differentiated cells (He et al., 2003).

Although the mechanisms explaining these heterogeneous phenotypes of virus-specific T cells are not known, there appears to be a correlation between the differentiation stage of virus-specific CD8 T cells and the control of viral replication mediated by the CD8 T cell response. CMV is a persisting virus with ongoing low-level viral replication, but it does not cause any disease in immune competent subjects; this is associated with high levels of fully differentiated effector CD8 T cells that control the ongoing replication of virus. On the other end of the spectrum, fluA does not persist in infected host. The fluA-specific memory T cells responsible for the long-term maintenance of immunity in healthy subjects are experiencing rest from stimulation by viral antigens (Wherry and Ahmed, 2004); this is associated with their early differentiation phenotypes. Between these two extremes lies EBV, which is a latent virus with periodic reactivation that re-stimulates the resting memory T cells briefly at intervals, resulting in an early-intermediate phenotype of the EBV-specific CD8 T cells. HIV causes a persistent and progressive infection. This is associated with an intermediate differentiation phenotype of HIV-specific CD8 T cells with certain functional defects when compared to CMV-specific CD8 T cells (Appay et al., 2000; Champagne et al., 2001). Such an immune response may partially control the ongoing viral replication during the asymptomatic phase, until the depletion of helper CD4 T cells leads to the collapse of the immune system and onset of AIDS.

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However, when HCV-specific CD8 T cells are compared to CD8 T cells specific for the other viruses, they do not fit into the general pattern described above. While HCV causes persistent infection and ongoing disease, HCV-specific CD8 T cells, if ever detected in the peripheral blood, are frequently found to have a phenotype consistent with an early differentiation stage, similar to the resting fluA-specific memory CD8 T cells which had not been exposed to the virus since the last acute influenza was cleared. Some of the HCV-specific CD8 T cells even appeared to be functional when stimulated ex vivo with the endogenous viral peptides of the patient (He et al., unpublished data). In other words, HCV-specific CD8 T cells in many patients appear to be in a state of rest or anergy in vivo, ignoring the ongoing HCV infection. Of particular interest is a recent study on a cohort of patients co-infected with HCV and CMV, which found that CMV-specific CD8 T cells in these patients appeared to have lost some markers associated with differentiation maturity, including increased expression of CCR7 and reduced expression of Fas and perforin, although they maintained functional responses to in vitro stimulation with CMV antigen (Lucas et al., 2004). The authors suggested that the reduction in mature CD8 T cells in HCV-infected individuals arises through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. In either case, HCV may have a pervasive influence on the general T cell immunity of infected hosts, which is not limited to HCV-specific T cells.

A critical factor for the development and differentiation of T cells into functional memory and effector cells is the stimulation that they receive during the primary response (Lanzavecchia and Sallusto, 2002). In order to understand the apparent weak or abnormal T cell immunity to HCV, it is important to investigate the initial events leading to the antiviral adaptive immunity, which is the processing and presentation of viral antigens.

DENDRITIC CELLS – ARE THEY INFECTED BY HCV?While B and T lymphocytes are the effectors of the adaptive immunity, their development and function is under the control of dendritic cells (DCs). Different subsets of DCs, including myeloid-derived DCs (MDC) and plasmacytoid-derived DCs (PDC), are the most important antigen presenting cells with the capability to capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate B and T cell responses (Banchereau et al., 2000). DCs not only activate lymphocytes, they also have the important function of tolerizing T cells to self-antigens, which results in the anergic state of such cells to avoid autoimmune reactions (Banchereau et al., 2000). The two opposite roles of DCs have been related to different maturity and cytokine production patterns of DCs (Crispe, 2003; Kubo et al., 2004). Thus, depending on the nature of antigens as foreign or self, the functions of DCs have to be precisely controlled to either activate or tolerize T cells. Any perturbation to this control

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may lead to serious consequences. These include impaired immunity to infecting pathogens, causing persistent infections; or abnormal immunity to self-antigens, causing autoimmune diseases. In addition to functioning as antigen presenting cells that play a key role in the induction of adaptive immunity or immune tolerance, DC is also an important part of the antiviral innate immunity which is largely mediated by the type I interferons (IFNs) produced by PDCs (Cella et al., 1999).

Many viruses can directly infect DCs through different cell surface receptors. In response to this invasion, DCs process viral proteins and present them through MHC class I and II pathways while undergoing a maturation that enhances their presentation of antigen to T cells and expression of T cell costimulation factors for induction of adaptive T cell antiviral immunity (Carbone and Heath, 2003; Rinaldo and Piazza, 2004). As a strategy to counteract antiviral immunity, some viruses have evolved mechanisms to undermine the functions of DCs. For example, infection of DCs by measles virus resulted in diminished IL-12 production and inhibition of DC maturation (Schneider-Schaulies et al., 2003; Servet-Delprat et al., 2003). HIV-infected subjects had defects in the number, immunophenotype and functions of blood DC subsets infected with HIV (Barron et al., 2003; Donaghy et al., 2003). Infection of DCs by CMV has also been shown to cause inhibition of DC maturation and T cell activation, as well as increased production of molecules that induce apoptosis in T cells and down-regulation of MHC class I molecules (Raftery et al., 2001; Moutaftsi et al., 2002).

Accumulating evidence suggests that DCs are susceptible to HCV infection. In some studies, HCV RNA sequences, including replicative negative-strand RNA, have been detected in DCs isolated from patients with chronic HCV infection (Bain et al., 2001; Goutagny et al., 2003; Tsubouchi et al., 2004a). It was reported that both immature and mature monocyte-derived DCs were infected with HCV, as indicated by detection of negative-strand HCV RNA in the cells incubated in vitro with HCV-positive serum samples (Navas et al., 2002). In a study of HCV RNA sequencies isolated from liver and DC samples of a patient, the quasispecies detected in DCs were unique and differed from those present in the liver, suggesting a particular tropism of HCV quasispecies for DCs. Moreover, the translational activity of DC-derived HCV was significantly impaired when compared with those from liver and PBMCs, suggesting an impaired replication of HCV in the DCs (Laporte et al., 2003). Infection of DCs by HCV is thought to be mediated by the interaction of the HCV glycoprotein E2 with DC-SIGN, a membrane-associated C-type lectin that also involves in the binding of HIV to DCs (Lozach et al., 2003; Pohlmann et al., 2003).

Several groups have reported dysfunction of DCs that may potentially affect adaptive immunity in patients with persistent HCV infection, using various in vitro

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functional assays for DCs. These include impaired allostimulatory abilities to CD4 T cells (Kanto et al., 1999; Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Kanto et al., 2004; Tsubouchi et al., 2004a), defects in responding to maturation stimuli (Auffermann-Gretzinger et al., 2001), as well as impaired ability to secrete IL-12, a cytokine important for the development of CD4 helper T cell responses (Anthony et al., 2004; Kanto et al., 2004). Some of these defects were reversed after IFN-α therapy that cleared HCV in the sera (Auffermann-Gretzinger et al., 2001) or DCs (Tsubouchi et al., 2004b), indicating that the DC dysfunction is associated with HCV infection. Interestingly, a positive association was observed between MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-infected subjects (Anthony et al., 2004). The DC-mediated innate antiviral immunity also appeared to be impaired in HCV-infected patients, as indicated by reduced production of IFN-α by PDCs (Anthony et al., 2004; Kanto et al., 2004). Of note, IFN-α is not only an important antiviral cytokine; it is also an important modulator for adaptive immunity. It has been reported that IFN-α enhances expression of class I and class II molecules, cytokines and perforin that are involved in the presentation of viral antigens and the effector functions of T cells (Ji et al., 2003), as well as provides a stimulating signal for the clonal expansion and differentiation of CD8 T cells (Curtsinger et al., 2005).

In addition to the dysfunction of DCs, the numbers of MDCs, PDCs and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy controls (Kanto et al., 2004; Wertheimer et al., 2004). Taken together, these findings point to a defective immune response mediated by HCV-induced DC dysfunction as a potential mechanism enabling the persistent HCV infection.

It should be mentioned that most of the reported DC dysfunctions were based on monocyte-derived DCs generated in vitro. While this system has been used extensively in studying DC biology, it is not clear to what extend this model represents the functions of DCs in vivo (Kanto and Hayashi, 2004), especially those in the infected liver. In addition, conflicting results have been reported by other investigators who had conducted similar studies but failed to detect monocyte-derived DC dysfunction in HCV chronically infected patients (Longman et al., 2004) or chimpanzees (Rollier et al., 2003; Larsson et al., 2004), the only animal model for HCV infection. Obviously, more studies are warranted to elucidate the exact role of DCs in the apparent HCV-specific immunodeficiency.

INTERFERENCE OF HOST CELL FUNCTIONS BY HCVWhile it is still controversial in terms of DC dysfunction in HCV-infected patients, in vitro studies have shown that various HCV proteins have the potential capability to modulate host cell functions by interfering with cellular signal transduction.

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Numerous interactions between HCV proteins and cellular components have been identified in cell lines or experimental mice by using different expression systems for HCV proteins (Tellinghuisen and Rice, 2002). Studies have showed that the expression of HCV proteins suppressed IFN-induced signal transduction through the JAK-STAT pathway (Heim et al., 1999; Blindenbacher et al., 2003; Geiss et al., 2003; Duong et al., 2004). Specifically, a recent study in transfected cell line demonstrate that expression of HCV proteins suppressed IFN signaling by degrading STAT1, a major signal protein of the JAK-STAT pathway (Lin et al., 2005). HCV NS3/4A serine protease blocks viral activation of IFN regulatory factor-3 (IRF-3), a key transcription factor in inducing type I IFN expression, by proteolytic cleavage of a cellular protein in the IRF-3 signaling pathway (Foy et al., 2003), while HCV NS5A and E2 inhibits the IFN-inducible protein kinase PKR thought to play a role in the antiviral effect of IFN (Gale et al., 1999; Taylor et al., 1999). In addition to their central role in the innate immunity against viral infections, type I IFNs also exert modulation functions to the adaptive antiviral immunity (Boehm et al., 1997; Foster, 1997; Ji et al., 2003; Diepolder, 2004; Curtsinger et al., 2005). Of particular interest, DCs are a key component of both innate immunity and adaptive immunity, which orchestrate a successful overall immune response against infecting viruses. In an intriguing in vivo mouse study, Sarobe et al. used recombinant adenovirus vectors encoding HCV core/E1 or NS3 proteins to demonstrate that expression of specific HCV proteins in DCs down-modulated the antiviral adaptive immunity (Sarobe et al., 2003). The authors found that the expression of core/E1 proteins in DCs inhibited their maturation. When mice were immunized with immature DCs transduced with an adenovirus encoding core/E1, lower CD4 and CD8 T cell responses were induced in comparison to the mice receiving DCs transduced with an adenovirus encoding NS3.

In addition to the effects of intracellularly expressed HCV proteins, the interference of cellular functions by extracellular HCV proteins binding to cell surface receptors has also been documented. CD81 is a cell surface marker that binds the major envelope protein E2 of HCV (Pileri et al., 1998), and has been suggested to be involved in the infection process of HCV (McKeating et al., 2004; Zhang et al., 2004). It was reported that engagement of CD81 with exogenous HCV E2 affected multiple functions of natural killer (NK) cells including activation, cytokine production, proliferation, and cytotoxic granule release (Crotta et al., 2002; Tseng and Klimpel, 2002). While the NK cell is a primary effector of the innate immune system, a complex interaction exists between NK cells and DCs (Andrews et al., 2005), which may lead to activation or killing of DCs, depending on the nature of the interaction (Ferlazzo et al., 2002; Gerosa et al., 2002; Moretta, 2002; Piccioli et al., 2002; Zitvogel, 2002). This indicates an important role of NK cells in the regulation of adaptive immunity to infections. It has been shown in a mouse model that NK cells are necessary for optimal priming of adenovirus-specific T cells (Liu

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et al., 2000). Of particular interest, in a study on the regulation of NK cell activities by inhibitory receptor CD94/NKG2A that normally leads to NK cell-induced activation of DCs, NK cells from chronic HCV-infected donors were not capable of activating DCs under the same conditions. In comparison to NK cells from normal donors, those from HCV-infected patients showed higher expression of the inhibitory receptor CD94/NKG2A and the cytokines IL-10 and TGF-β (Jinushi et al., 2004). Therefore, modulation of NK cells could be another potential pathway for HCV to affect the host innate and adaptive immune responses.

While E2 is a major component of HCV envelope and is readily available in HCV-positive serum for interaction with cell surface receptors, it was reported that free HCV core protein was secreted from stable transfectant cell lines (Sabile et al., 1999) and could be detected in the serum of HCV-infected patients as well (Maillard et al., 2001). In a series of publications, Hahn and colleagues reported in vivo and in vitro studies suggesting that HCV core acts as an immunomodulator for the host T cell response. They first demonstrated that the core protein of HCV genotype 1a delivered by a recombinant vaccinia vector suppressed the immune response to the vaccinia virus in mice (Large et al., 1999). Using in vitro cell systems, they further showed that the core protein bound to the complement receptor gC1qR on T cells and inhibited their proliferation and IFN-γ production (Kittlesen et al., 2000; Yao et al., 2001; Yao et al., 2003; Yao et al., 2004). Based on these results, a model has been proposed that HCV core acts as an immune modulator that binds to a component of the host complement system and suppresses T cell responses, leading to the persistence of HCV (Eisen-Vandervelde et al., 2004). This is an attractive model because the binding of C1q, the natural ligand for gC1qR, to T cells is already known to suppress T cell response (Chen et al., 1994). In addition, other pathogens, including measles virus, EBV, and HIV, also appear to exploit similar strategies to suppress the host immune system by interactions with components of the host complement machinery (Fingeroth et al., 1984; Viscidi et al., 1989; Karp et al., 1996).

However, the role of HCV core as an immunomodulator is still an issue of debate, as similar studies using the core of HCV genotype 1b delivered with a recombinant adenovirus vector, or using genotype 1b core transgenic mouse, both failed to demonstrate any immunomodulatory effects on virus-induced cellular immunity (Sun et al., 2001; Liu et al., 2002). If the immunomodulator function of HCV core is a unique feature of HCV genotype 1a but not genotype 1b, this does not explain the fact that both HCV strains are equally likely to establish persistent infection. Therefore, this model needs direct evidence from human clinical studies, as well as more vigorous testing with in vivo experimental systems, including chimpanzees.

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REGULATORY T CELLS IN HCV INFECTIONRegulatory T cells (Tregs) have been recognized to be an important modulator of T cell immunity in recent years. The most studied Tregs are those with the phenotype CD4+CD25+, which have been shown to be powerful inhibitors of T cell activation both in vivo and in vitro (Shevach, 2002). While the involvement of Tregs in human autoimmune diseases such as multiple sclerosis and myasthenia gravis has been established (Viglietta et al., 2004; Balandina et al., 2005), the potential role of Tregs in viral hepatitis is just beginning to be defined. They could limit liver injury by controlling inflammation, or they may promote persistence of infection by suppressing immune responses (Chang, 2005). Recently, increased numbers of these cells have been linked to the impaired immune response in patients with chronic HBV infection (Stoop et al., 2005). In chronic HCV infection, the persistence of HCV was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequencies of CD4+CD25+ Tregs that could directly suppress HCV-specific CD8 T cells ex vivo (Sugimoto et al., 2003; Cabrera et al., 2004). However, these studies did not answer the question of whether the abnormalities of Tregs associated with persistent HCV infection are the causes or the consequences of chronic HCV infection.

Studies in mice have linked the immune regulatory function of Tregs to DCs (Pasare and Medzhitov, 2003). It was reported recently that the suppressive function of Tregs was critically dependent on immature DCs and was readily reversed by the maturation of DCs (Kubo et al., 2004), indicating that the maturity of DCs is a key factor that determines suppression or activation of adaptive immune response. Therefore, if the functions of DCs are indeed modulated by HCV infection, Tregs may provide another potential pathway for HCV to manipulate host adaptive immunity to benefit its persistence.

IMMUNE CELLS AND IMMUNE RESPONSES IN THE LIVERFig. 1 is a summary of the aforementioned potential mechanisms for the regulation of adaptive immunity by HCV. It should be emphasized that these models are largely based on in vitro or ex vivo experiments using human immune cells isolated from peripheral blood or on mouse experiments and have not been verified in HCV infected patients. A major challenge to all these potential mechanisms, or the HCV-induced dysfunctions of different immune cell subsets including DCs, NKs and T cells, is that they are not in agreement with the lack of global immune deficiency in HCV-infected individuals similar to that in HIV-infected individuals. In other words, these mechanisms cannot explain why only HCV-specific immune responses are impaired, while the immune responses against other pathogens appear to be spared from the HCV-mediated immune dysfunctions. Although a recent study suggested that in patients with chronic HCV infection the phenotypes of CMV-specific CD8 T cells were affected, there was no evidence for functional defects of CMV immunity (Lucas et al., 2004).

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The primary site of HCV replication and the major location of disease caused by HCV are both in the liver, where most of the immunopathologic events associated with the infection are likely to occur. This notion is supported by the dramatic lymphocyte infi ltration in the infl amed liver, but not in the normal liver. Unfortunately, because of the diffi culty in obtaining liver specimens, most of the immunological studies on human liver diseases have to rely on peripheral blood samples. Although very little is known about the immune cells in human liver compared to their counterparts in the peripheral blood, evidences derived from limited studies that directly investigated immune cells in normal and HCV-infected livers have revealed signifi cant differences between the intrahepatic and peripheral lymphocyte subsets, including their activation status, phenotypes and proliferation capability (Nuti et al., 1998; Wang et al., 2004; Ward et al., 2004). By using MHC tetramers, HCV-specifi c CD8 T cells in the liver have been characterized directly. These studies revealed that such cells were enriched in HCV-infected liver versus peripheral blood and had different surface phenotypes compared to their counterparts in the periphery (He et al., 1999; Grabowska et al., 2001; Accapezzato et al., 2004). Of note, studies in mice have demonstrated a highly heterogeneous nature of hepatic DCs and identifi ed unique intrahepatic DC subsets with phenotypic and functional features distinct from DC subsets isolated from other sites (Lian et al., 2003).

Fig. 1. Potential mechanisms for HCV-mediated interference of adaptive immunity. All these mechanisms are likely to operate primarily in the liver.

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As the largest organ in the body, the liver not only has various excretory, detoxifying and metabolic functions, but it is also considered an intrinsic lymphoid organ (Mackay, 2002) with unique microenvironment compared to other lymphoid tissues in the periphery, such as the lymph nodes. Therefore, naive T cells in the liver may encounter local antigens and start development and differentiation in a manner distinct from those in the periphery, including such dramatic differences as apoptosis versus proliferation (Park et al., 2002). It has been shown in mice that oral administration of a foreign antigen at high dosage generated CD4 Tregs that suppressed T cell proliferation as well as Ab responses to the antigen (Watanabe et al., 2002). Of particular interest, Bowen et al. recently demonstrated in mice that the site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. They showed that while naive CD8 T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury (Bowen et al., 2004). These findings emphasized the unique nature of immune responses in the liver versus the periphery.

The intrahepatic tolerance can be considered a requirement for the special functions of the liver. The incoming blood stream from the intestine to the liver carries large amount of food-derived antigens that are foreign in nature but mainly harmless to the body. The constant presence of non-self antigens in the liver is thought to impose a constraint on the immune responses generated in the liver, resulting in a tolerant environment for foreign antigens (Crispe, 2003). The unique tolerance nature of liver is best demonstrated by the fact that allogeneic liver transplantation can be established without immunosuppression (Calne et al., 1969). However, this constraint on liver immunity does not prevent the immune system from mounting vigorous responses against some liver-specific pathogens such as hepatitis A virus, which is almost always cleared after a self-limited infection (Hollinger and Emerson, 2001), and HBV, which is also cleared in more than 90% of immunocompetent adults (Hollinger and Liang, 2001). Obviously, a precise control on the actions of the intrahepatic immune cells is in operation, leading to either tolerance or immunity to foreign antigens.

Although the controlling mechanism for liver tolerance is poorly understood at this time, it is reasonable to speculate that liver resident antigen presenting cells, including DCs, liver sinusoidal endothelial cells (Knolle and Limmer, 2001) and liver resident machrophages or Kupffer cells (Everett et al., 2003), play a critical role in shaping the outcome of intrahepatic immune responses. In addition to the professional antigen presenting cells, hepatocytes may also serve as antigen presenting cells under certain conditions (Herkel et al., 2003). It has been shown in mice that liver sinusoidal endothelial cells selectively suppressed the expansion of

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IFN-γ-producing Th1 cells but promoted the outgrowth of IL-4-expressing Th2 cells, creating an immune suppressive milieu that favors development of tolerance rather than immunity within the liver (Klugewitz et al., 2002). In particular, the accessory signals delivered by the hepatic antigen presenting cells, including cytokines and costimulating molecules, are likely to exert profound effects on the regulation of intrahepatic T cell immunity (Crispe, 2003). Given that liver is the primary site of replication for HCV with the highest concentration of viral protein products, it is conceivable that HCV-mediated interference of immune cell functions (Fig. 1), including those of HCV-infected DCs or other antigen presenting cells as well as NK cells and T cells, occurs primarily in the liver rather than other sites of the body, resulting in a suppressed immunity against HCV but relatively unaffected immune responses against other pathogens that do not primarily infect the liver. Future studies on the issue of HCV-mediated immune modulation should focus on the relevant events in the liver that affect the function of intrahepatic immune cells.

CONCLUSIONAlthough the mechanism for HCV to evade host immune responses and establish chronic infection is still poorly understood, accumulating data indicate that HCV may play an active role in attenuating host adaptive immunity to benefit its persistence. Therefore, suppression of HCV replication by antiviral treatment should restore the T cell immunity against HCV. Indeed, in HCV chronically-infected patients treated with IFN, increased T cell immunity after IFN therapy has been demonstrated for HCV-specific CD4 T cells (Cramp et al., 2000; Barnes et al., 2002; Kamal et al., 2002) and CD8 T cells (Vertuani et al., 2002; Morishima et al., 2003), although in some studies an increase in HCV-specific CD8 T cells was not detected (Barnes et al., 2002). The discrepancy could be caused by different methods and antigens used to measure CD8 response and should be resolved by further studies.

While current pegylated IFN-based anti-HCV therapies have accomplished greatly improved efficacy, the rate of sustained virological response is still less than 50% for the most common genotype of HCV (Manns et al., 2001; Fried et al., 2002). In a significant fraction of patients who failed to achieve long-term response, however, the HCV replication was temporally suppressed during IFN treatment. This may represent an opportunity for immunotherapy such as therapeutic vaccines designed to enhance adaptive immunity against HCV. With the HCV viral load suppressed by IFN, the therapeutic vaccine is likely to elicit an anti-HCV immunity most efficiently. Therefore a combination of antiviral drugs and a therapeutic vaccine may produce a synergetic effect that surpasses the potential of either treatment strategy alone.

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HCV Vaccines and Recombinant VSV

Chapter 15

Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy

Ayaz M. Majid and Glen N. Barber

ABSTRACTSeveral vaccine strategies have been attempted in chimpanzee and smaller animal models to generate immune responses to hepatitis C virus (HCV). While neutralizing antibody may play a role in preventing HCV infection, studies in chimpanzees and humans during rare cases of acute resolving HCV infection indicate that, HCV immunity appears to be associated with vigorous, sustained and multi-specific Th1 intra hepatic CD8+ and CD4+ T cell responses. Several new promising technologies utilizing viral based vaccine approaches that appear to generate both antibody and cell mediated immune responses have recently been reported. These include viral vectors that express HCV products and non-replicating viral like particles (VLPs) that appear to induce T-helper type 1 (Th1) immune responses considered important in resolving HCV infection. In addition, viral vectors based on recombinant vesicular stomatitis virus (rVSV) may offer safe yet potent stimulation of both innate and adaptive immune responses. Here, we review the successful application of viral based vaccines, including VSV in generating viral immunity in animal models and describe the potential usefulness of this technology as a strategy for HCV vaccine design and immunotherapy.

INTRODUCTIONThe inhibition of virus replication by the immune system is of paramount importance to limit the spread of infection and moderate the course of the disease. Essentially, control of viral infections consists of non-specific innate immune responses and adaptive responses to viral specific proteins (Parkin and Cohen, 2001). Vaccine intervention aims to stimulate B cell antibody production (humoral) and cell mediated (CD4+ and CD8+) immunity (Begue, 2001b). However, there have been several major obstacles that have hampered the development of an effective HCV vaccine. Firstly, apart from humans, the only infectious HCV animal model is the chimpanzee, a protected species that is costly and hence limited in its availability (Bukh, 2004). Secondly, although extensive studies from chimpanzees and patients have provided insights into immune responses, it remains

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unclear as to why the immune system, in many cases, is inefficient in eliminating HCV infection (Gremion and Cerny, 2005). Despite detectable humoral and cell mediated immunity, HCV induces 50-80% chronicity of infected individuals leading potentially to cirrhosis and hepatocellular carcinoma (Harris et al., 2002). HCV is also highly variable, divided into at least 6 major genotypes and more than 50 subtypes based on nucleotide diversity within core, E1 and NS5 genes (Zein, 2000). In addition, as observed with other RNA viruses, HCV exists as groups of related but distinct viral populations termed quasispecies variants that differ in sequence diversity within distinct hypervariable regions along the genome. The N-terminus of the E2 glycoprotein (a prime vaccine target) contains a major hypervariable region 1 (HVR-1) and sequence variation in the HVR1 has been associated with immunologically driven HVR1-antibody escape mutants (Farci et al., 1997; Majid et al., 1999). The first neutralizing antibody epitopes were located within the HVR-1 region, and hyperimmune serum obtained from immunization of a rabbit with HVR-1 peptide has been found to protect against homologous HCV in chimpanzees, but did not protect against 'escape' mutants that persisted during chronic infection (Farci et al., 1996). Nevertheless, studies associated with intravenous drug users who resolved previous HCV infection and who are less likely to be re-infected suggests that immunity against HCV can be successfully generated in some individuals (Mehta et al., 2002). Furthermore, recent work illustrates that targeting multiple epitopes in the HCV envelope proteins (E1 and E2) may facilitate a more broad neutralization capacity (Bartosch et al., 2003a). Immunological studies of acute phase HCV infection suggest that this period is critical for determining the outcome of infection (Thimme et al., 2001). In humans and chimpanzees, the clearance of acute infection is accompanied by a strong and multi-specific CD4+ and CD8+ T cell response (Haefelin-Neumann et al., 2005; Rehermann and Nascimbeni, 2005). Immune mediated control of HCV infection is also evident from intrahepatic compartmentalization of HCV specific T cells, aggressive disease progression in HCV/HIV co-infected individuals and increased viral loads with immunosuppression of patients (Gremion and Cerny, 2005).

Here, we examine the problems encountered in the development of HCV vaccines and evaluate current as well as future vaccine strategies to generate effective immune responses to HCV. Several approaches designed to elicit immune responses to HCV structural proteins (Core, E1 and E2 glycoproteins) and the bi-functional viral serine protease/helicase (NS3) that contains conserved 'immunodominant' regions, have been tested in non-human primates, monkeys and murine models. Although a comprehensive review of these technologies is beyond the scope of this chapter, we briefly describe the need for more optimal approaches to HCV vaccine design. In this regard, we discuss the recombinant Vesicular Stomatitis Virus (VSV) as a vector that could be useful in the fight against HCV infection.

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CLASSICAL STRATEGIES IN VIRAL VACCINES The principle of vaccination is to induce a 'primed' state in the host so that infection with a pathogen will result in a rapid secondary immune response. The goal to eliminate or inhibit replication of the organism and protect from clinical disease is dependent upon memory T and B cells as well as neutralizing antibody in the serum. Virus infection and replication in host cells is known to elicit long-lived antibody and cell-mediated immunity. These features make viral vectors attractive as vaccine candidates after safety issues are addressed by attenuation or inactivation, since they can often induce long-term immunity following a single dose.

Non-virulent viral vaccine strategies have been successfully developed for a number of viral pathogens that can be grown in cell culture to facilitate their attenuation or inactivation (Begue, 2001b, Gershon, 1990; Hinman and Orenstein, 1990; Matter, 1997). Live attenuated viruses typically have reduced virulence caused by repetitive passage during in vitro cell culture growth conditions. Selected mutants replicate poorly in the host and do not cause disease but efficiently induce long-lived antibody and cell-mediated immunity. Indeed, Measles, Mumps and Rubella (MMR vaccine) are controlled in many developed countries through this live attenuated vaccine approach (Burgess, 1994; Wharton et al., 1990; Zimmerman and Burns, 1994). The worldwide eradication of smallpox is another example of a live attenuated heterologous vaccine. In this case, the cross reacting immunity of vaccinia (less virulent) is protective against variola virus, the causative agent of small pox (Begue, 2001a, Enders et al., 2002). Selectively targeted live attenuated vaccines also include single doses of yellow fever for travellers and varicella-zoster virus for the elderly (Hill, 1992; Marfin et al., 2005; Senterre, 2004; Takahashi, 2004). The main drawback of live attenuated vaccines however, is the danger of reversion to virulence and the possibility of causing extensive disease in immunocompromised individuals.

When live attenuated vaccines are unavailable, inactivated preparations of the virulent organism using beta-propiolactone or formaldeheyde are an option (Bachmann et al., 1993; Jiang et al., 1986; King, 1991). However, these inactivated vaccines generally only stimulate humoral responses, are expensive to prepare and in addition, the chemical inactivation can directly impair certain immune responses such as T cell activation (Bachmann et al., 1993). However, the Salk poliovirus vaccine containing all 3 polio-virus strains is a successful example of this approach and is particularly useful in protecting immuno-suppressed children (Pearce, 2004). The live, less expensive Sabin polio-vaccine has been adopted in many parts of the world due to lower costs and elicits effective induction of mucosal immunity (Pearce, 2004). Seasonal Influenza vaccines similarly comprise inactivated Influenza A and B strains (Hill, 1992; Marfin et al., 2005; Schwartz and Gellin, 2005; Takahashi, 2004). For Rabies virus, a human diploid cell culture-derived inactivated vaccine

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is administered either for post exposure prophylaxis following a rabid animal bite or pre-exposure prophylaxis to protect animal workers at risk of infection from occupatioal exposure (Lodmell and Ewalt, 2004).

In regard to other major etiological agents of liver disease, effective hepatitis A virus formalin inactivated cell culture vaccines are available (Provost et al., 1986; Strader and Seeff, 1996). Two doses administered one month apart appear to induce high levels of neutralizing antibodies. For hepatitis B virus (HBV), HBV surface protein purified from viral carriers, or a recombinant viral approach have been successfully utilized to protect against HBV infection (Coursaget et al., 1990; Goilav and Piot, 1989; Magnani et al., 1989; Prince et al., 1984; Szmuness, 1979). In the first strategy, a trial in homo-sexual men in the USA showed that 3 intramuscular injections at 0, 1 and 6 months appeared to protect 95% of vaccinees (Goilav and Piot, 1989). However, the latter HBV vaccine is now more widely used in the universal childhood immunization scheme and is given at 6, 10, and 14 weeks of age, and likely requires a booster later in life (Milne et al., 1992; Miskovsky et al., 1991; Murata et al., 1989). Importantly, HAV and HBV vaccination demonstrates the plausibility of protection against hepato-pathogens that replicate primarily in immune-compromised environment of the liver (Willberg et al., 2003). However, it has been more difficult to develop HCV vaccine along similar lines since HCV does not replicate efficiently in cell culture. Recently, three independent reports (see chapter 16) describe complete HCV replication of a chimeric genotype 2a replicon in cell culture, which may facilitate vaccine strategies (Lindenbach et al., 2005). Thus, large scale purification of chemically inactivated or attenuated HCV strains using this technology remains an exciting prospect for HCV vaccine studies. Meanwhile, alternative recombinant approaches to engineer immune responses to HCV have currently been used in HCV vaccine studies.

STUDIES OF RECOMBINANT HCV PROTEIN SUB-UNITS IN CHIMPANZEESIn the early 1990s, chimpanzees were immunized with purified recombinant HCV E1 and E2 glycoproteins as these were presumed targets for virus neutralization. The source vectors for these subunit vaccines were recombinant vaccinia (rVV) since rVV expressed products were known to elicit potent antibody responses in alternate vaccine regimens (Choo et al., 1994; Ralston et al., 1993). These rVV vectors demonstrated high-level protein expression from prototype HCV-1 structural (core-E1-E2 1-906) cDNA (Ralston et al., 1993). Purified E1 and E2 products mixed with the MF59 adjuvant also produced neutralizing antibodies since protection was observed following challenge with low doses of homologous HCV but not against heterologous HCV infection (Choo et al., 1994; Houghton et al., 1997; Ott et al., 1995). Subsequently, since E1/E2 antigens appeared susceptible to genetic variation amongst HCV types, the HCV core protein that is highly conserved

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amongst HCV genotypes was also evaluated. However, since subunit vaccines alone appear inefficient at inducing cytotoxic T lymphocyte (CTL) responses, the core protein was combined with a 40nm matrix composed of saponins, cholesterol and phospholipids (ISCOM) (Polakos et al., 2001). The classical ISCOM method entraps the antigen inside the adjuvant platform to facilitate priming of CD4+ and CD8+ mediated responses (Takahashi et al., 1990). For HCV vaccine studies, a non-classical ISCOM approach was used where E. coli purified core protein was adsorbed onto ISCOMATRIX. The resulting particulates stimulated strong long-lived, CD4+ and CD8+ responses and induced Th0-type (Th1 and Th2-type cytokines) as well as anti-core antibodies in Rhesus Macaques (Polakos et al., 2001). The prospects of HCV ISCOM-vaccines to produce sterilizing immunity awaits to be reported but a core vaccine itself may have therapeutic value since core-specific CTLs in HLA-B44+ patients co-incided with lower viral titers, and core-CD4+ T cell responses correlated with milder courses of liver disease (Bottarelli et al., 1993; Hiroishi et al., 2004)

To faciliate broader responses to HCV types, other studies have utilized truncated forms of E1aa192-330 and E2aa 390-683 (HCV-N2) purified from baculovirus-infected cells combined with HVR-1 peptides from different isolates (HCV-6) (Esumi et al., 1999). However, despite high antibody responses to E1/E2 in chimpanzees, the low level immune responses to HVR-1 peptides resulted in lack of sterilizing immunity to HCV-6 challenge that was achieved only by boosting HVR-1 (HCV-6) antibody responses. It seemed that antibody responses alone were incapable of neutralizing HCV infection and these observations pointed to the requirement for technologies that may facilitate broader immune responses to control HCV infection. In this regard, the introduction of DNA vaccination technologies offered alternative or complementary approaches to E1/E2 subunit vaccines.

EFFICACY OF RECOMBINANT DNA APPROACHES IN GENERATING HCV IMMUNITYNaked DNA or plasmid vaccines encode viral genes that following inoculation of a host are expressed to stimulate immune responses. DNA vaccination was reported to prime good antibody responses and offer the advantage of increasing CTL responses (Donnelly et al., 1997). In fact, DNA-derived immunogens encoding HCV core and E2 sequences alone or fused with hepatitis B surface antigen induced antibody and CTL responses to HCV or HBV in BALB/c mice (Inchauspe et al., 1997). However, the recombinant DNA-vaccinated animals lacked neutralizing antibodies that could block binding of purified HCV E2 to the putative cellular receptor, CD81 (Heile et al., 2000; Pileri et al., 1998). Furthermore, the same study highlighted that endoplasmic retained recombinant E2 protein expressed in mammalian cells was superior in eliciting neutralizing antibodies. The latter finding stresses the importance of generating antigens that are correctly proteolytically processed and therefore authentically folded (discussed below).

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Combining DNA vaccination with boosts of purified protein or recombinant viruses expressing the same antigens appear to enhance immune responses (Pancholi et al., 2000; Pancholi et al., 2003; Song et al., 2000). However, in order to assess the effectiveness of immune responses generated using these and other HCV vaccine approaches, surrogate HCV challenge technologies have been created to replace the need for expensive chimpanzee models in preliminary studies. Two examples include: vaccinia-expressing HCV structural proteins, or Listeria monocytogenes-expressing HCV NS3, both of which were developed to monitor the efficacy of elicited immune responses in murine and potentially higher animal models (Pancholi et al., 2003; Simon et al., 2003). The HLA-A2.1 transgenic mouse model has been especially useful for refining cell-mediated HCV immunity (Pascolo et al., 1997). These mice are devoid of murine MHC-I molecules and are transgenic for human HLA-A2.1 (A0201) monochain major histocompatability class I molecule. Studies have shown that these mice recognized the same HCV-derived peptides found in human HLA-A2.1-restricted CTLs (Arichi et al., 2000; Brinster et al., 2001). DNA vaccines can be positively modulated as shown in murine studies with adjuvants such as CpG motifs and Quil A that appear to induce Th1-(IL2 and IFNγ cytokine profile) biased immune responses in addition to strong antibody responses as demonstrated in DNA vaccination to HCV NS3 (Hong et al., 2004). Furthermore, efficient presentation and priming of cell-mediated responses can be optimized using cationic microparticles that carry DNA-based vaccines (Hagan et al., 2004). Alternatively, recombinant semliki forest virus (rSFV) particles expressing NS3, in combination with DNA vaccination or alone, have been found to stimulate strong immune responses in mice (Brinster et al., 2002). The rSFV particles infect host cells but do not replicate. However they express high levels of NS3, which appear to induce (as with DNA vaccine) NS3-specific CTLs targeted to dominant HLA-A2 epitopes described in patients (Urbani et al., 2001). However, only rSFV-NS3/DNA combination experiments elicited anti-NS3 antibodies in non-transgenic BALB/c mice (Brinster et al., 2002). Unfortunately, in chimpanzees, DNA immunization-encoding HCV E2 appeared incapable of generating sufficient antibody and cellular immune responses to clear HCV infection (Xavier et al., 2000). A good HCV vaccine should elicit strong antibody and cellular immune responses., and current studies in HCV-DNA vaccine approaches suggest combinatory vaccine strategies are likely required to enhance HCV antigen responses.

Advancements in recombinant viral technologies have led to additional strategies that may generate safer approaches to viral based vaccine regimens. These include production of virus-like particles (VLPs) that closely resemble properties of the native virion. These VLPs have been shown to elicit potent humoral and importantly cellular (CTL) activity as demonstrated with human papillomavirus-VLPs, recombinant HIV and HBV VLPs in animal studies (Kahn et al., 2001; Roberts et al., 1999; Roberts et al., 1998; Roberts et al., 2004; Rose et al., 2001). Recently,

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HCV-like particles based on C, E1 and E2 that are capable of stimulating CTL and humoral activity have also been reported (see discussion below).

HEPATITIS C VIRUS-LIKE PARTICLESRecombinant baculovirus expression of the HCV structural proteins (core/E1/E2) has been reported to generate HCV-like particles (HCV-VLPs) that have biophysical, ultrastructural, and antigenic properties similar to those of the putative virions (Baumert et al., 1998; Baumert et al., 1999). Immunization of BALB/c mouse strains or human HLA-A2 transgenic (AAD) mice with these 40-50nm non-infectious HCV-VLPs suggests that they are efficient at generating broad and vigorous humoral and cellular immune responses compared to immunization with DNA (Murata et al., 2003). Mice vaccinated with HCV-VLPs developed antibodies to HCV E1/E2. These anti-HCV E1/E2 antibody responses were enhanced by HCV-VLP plus monophosphoryl lipid A [MPL] and QS21 adjuvant (AS01B) or CpG 10105 and especially with combination of AS01B plus CpG 10105 (Qiao et al., 2003). Furthermore, isotype analysis of the induced anti-HCV envelope proteins demonstrated that HCV-VLP alone induced immunoglobulin (Ig) G1 response while the use of adjuvants ASO1B and CpG 10105 combined facilitated predominately IgG2a response that is indicative of a Th1 response proposed to be important for HCV clearance. The neutralization capacity of these anti-E1/E2 antibodies induced by HCV-VLP cannot be tested easily due to the lack of a suitable small-animal model system. However, by challenging mice with recombinant vaccinia expressing HCV structural antigens (vv.HCV.S-genotype 1b), investigators were able to examine the induction of immune protection in murine and higher animal models (Jeong et al., 2004; Murata et al., 2003). Although vvHCV.S infection is not representative of natural HCV infection, studies analyzing immunization strategies with baculovirus generated HCV-VLPs showed that HCV-VLP vaccinated mice were better protected against vvHCV.S than DNA immunization (Murata et al., 2003). This protection is due in part to the fact that, as compared with animals immunized with DNA methods, HCV-VLPs elicit strong CTL responses and vigorous CD8+ T cell responses against HCV core and E2 proteins. It appears that unlike recombinant protein subunit vaccines, these virion like structures can possibly be processed efficiently through the major histocompatibility complex 1 pathway and subsequently effectively prime CD8+ responses. HCV-VLPs therefore offer promise for further study in chimpanzee or human trials.

Recently, infectious particle systems have also been described where pseudo-particles are assembled that display unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles (Bartosch et al., 2003b, Flint et al., 2004). These particles were primarily designed to understand HCV cell entry but could provide useful information for vaccine development especially with regard to the potential neutralization of HCV glycoproteins to their target receptors using

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anti-sera of animals treated with different vaccine regimens. In fact, the presence of a green fluoresent protein marker packaged within these HCV-pseudotype allowed determination of infectivity mediated by the HCV glycoproteins in primary hepatocytes and hepato-carcinoma cells (Bartosch et al., 2003b). This infectivity was neutralized using patient sera and by some anti-E2 monoclonal antibodies, indicating a role for neutralizing antibodies against HCV glycoproteins. The potential modification of these particles to render them non-infectious may allow for in vivo vaccine studies in animal models.

In addition to the above studies, our laboratory and others have focused on using vesicular stomatitis virus (VSV) as a candidate for evaluation as a virus-based strategy for HCV vaccination and/or immuntherapeutic studies (Ezelle et al., 2002; Majid et al., 2005). The advantages of using VSV-based approaches to generate immune responses against HCV are discussed below.

VSV AS A SAFE AND POTENT VECTOR FOR GENERATING IMMUNITY AGAINST VIRAL PATHOGENSVSV is a member of the Rhabdoviridae family and is a negative-stranded cytopathic virus. Rhabdoviruses are classed into at least 5 genera. Vesiculoviruses, Lyssaviruses and Ephemeroviruses infect animals (both vertebrates and invertebrates), whereas Cytorhabdoviruses and Nucleorhabdoviruses infect plants. VSV is composed of an 11-kilobase negative-sense RNA genome and is relatively simple since it encodes for only five viral proteins: the nucleocapsid (N) that encases the virus genome, 2 polymerase proteins (L and P), a single surface glycoprotein (G) and a peripheral matrix protein (M). Rhabdoviruses are enveloped viruses and their glycoproteins are classed as typical type 1 membrane proteins consisting of polypeptide trimers of the viral G protein (Coll, 1995). This G protein is a dominant viral antigen and is responsible for viral infection through unidentified cellular receptor(s) on a variety of mammalian and insect cells, and has been shown to be a target for neutralizing antibody (Beebe and Cooper, 1981; Chan et al., 1982; Hardgrave et al., 1993; Lefrancois and Lyles, 1983).

There are several features that make VSV an excellent candidate as a vaccine vector. This weak human pathogen does not undergo genetic recombination or genomic reassortment and has no known transforming properties. Furthermore, VSV does not integrate any of its genomic material into host cell DNA (Barber, 2004; Lawson et al., 1995; McKenna et al., 2003). As a vaccine strategy, VSV is known to elicit strong humoral and cellular immune responses in vivo and naturally infects at mucosal surfaces (Haglund et al., 2002; Martinez et al., 2004; McKenna et al., 2003). This feature offers an alternative less invasive intranasal route of immunization that has been shown to induce both mucosal and systemic immunity (Kahn et al., 2001; Reuter et al., 2002; Roberts et al., 1999). In addition,

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recombinant VSVs can be generated to accommodate large foreign gene inserts or multiple genes into their genomes (Ezelle et al., 2002; Fernandez et al., 2002; Obuchi et al., 2003). Other RNA viral vectors, such as those based on alphaviruses and poliovirus, do not typically tolerate incorporation of large foreign genes to form replication-competent RNA viruses (Falkner and Holzer, 2004; Schlesinger, 2001). Furthermore, VSV grows to high titers in vitro, thus facilitating rapid purification of large amounts of virus and viral proteins.

However, natural VSV infection has been reported in cattle, horses and swine causing significant disease, including vesicular lesions around the mouth, hoofs, and teats with loss of beef and milk production (Martinez and Wertz, 2005). In the US, livestock is periodically infected with one of two serotypes of VSV (Indiana, VSVI or New Jersey, VSVNJ). VSV infection is asymptomatic in humans but in rare cases, chills, myalgia and nausea have been reported (Coll, 1995; Fields and Hawkins, 1967; Johnson et al., 1966; Wagner, 1996). As a consequence of rare infectivity in humans, seroprevalence of VSV antibodies within the general population is low except in limited regions in Georgia (VSV NJ ) or Central America (VSV I and VSV NJ ). Antibodies are also detected in individuals who have high risk of exposure such as laboratory workers, veterinarians and ranchers (Johnson et al., 1966). This low VSV seropositivity in the general population and the lack of serious pathogenicity in humans are advantages in the potential use of live recombinant VSV-vectored vaccines in humans. VSV-HCV PSEUDOTYPES AS A PRELUDE TO RECOMBINANT VSV IN HCV IMMUNO-INTERVENTIONSince VSV was known to infect many cell types and can incorporate foreign viral glycoproteins on its surface, it was used first in HCV research as a tool to generate reagents to understand HCV infection in cell culture. The use of VSV in HCV research initially focused in generating pseudotype viruses expressing HCV envelope proteins on their surface (Matsuura et al., 2001; Meyer et al., 2000). These recombinant VSV pseudotypes were used to understand HCV cell entry in the absence of conventional cell culture systems. Several lines of evidence using VSV-HCV pseudotypes pointed to functional role for both E1 and E2 glycoproteins in pseudotype virus infectivity. First, sera derived from chimpanzees immunized with homologous HCV envelope glycoproteins were found to neutralize virus infectivity. Secondly, as with many pH-dependent enveloped viruses, VSV-HCV pseudotype entry was negatively influenced by low pH pre-treatment in a number of susceptible cell lines (Meyer et al., 2000). The same study also demonstrated that Concavalin A (plant lectin) neutralized both E1 and E2 pseudotype virus infectivity. These findings illustrated that carbohydrate structures on HCV envelope glycoproteins may influence binding of HCV to cells directly or by attachment to carbohydrate binding proteins for attachment to or infection of cells. Furthermore, the use of monoclonal

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antibodies to block putative HCV cell entry receptors, CD81 or LDL, appeared to reduce pseudotype plaque titers demonstrating specificity of VSV-HCV pseudotype binding to cells (Agnello et al., 1999; Cormier et al., 2004; Matsuura et al., 2001; Meyer et al., 2000; Pileri et al., 1998). However, technical limitations in generating VSV-HCV pseudotypes resulted in expression of either HCV E1 or E2 alone, and not as a functional E1/E2 non-covalently linked glycoprotein complex as found in natural HCV infection (Beek et al., 2004). Furthermore, the chimeric nature of the recombinant HCV E1 or E2 fused to the cytoplasmic tail of VSV glycoprotein may influence interpretation of the results (Meyer et al., 2000). However, others also showed that VSV-HCV pseudotypes could be generated that possesed chimeric E1 or E2 glycoproteins either individually or together (Matsuura et al., 2001). In their report, VSV glycoprotein was replaced with the green fluorescent protein (GFP) and infectivity of pseudotypes was determined by GFP-expressing cells. Importantly, their study illustrated that co-expression of both HCV glycoproteins in the VSV-pseudotypes was required for maximal infectivity. Subsequently, we and others adapted an alternative VSV approach that utilized the genetic manipulation of the full length VSV genome to generate novel reagents for vaccine strategies.

REPLICATION COMPETENT AND DEFECTIVE RECOMBINANT VSV AS A VACCINE STRATEGY TO GENERATE IMMUNE RESPONSES The availability of recombinant DNA technology has allowed for genetic manipulation of the VSV genome and recovery of infectious VSV entirely from cDNA clones (Lawson et al., 1995). Rabies virus was the first Rhabdovirus to be recovered from a complete cDNA clone (Schnell et al., 1994). An important quality of the approach utilized the initiation of the infectious cycle by expressing the antigenomic RNA rather than the genomic RNA in cells expressing the viral N, P, and L proteins. This strategy avoids potential anti-sense problems effecting viral replication in which mRNAs encoding the N, P, and L proteins would hybridize to the negative-strand genomic RNA. This same approach has been successfully applied to full-length positive-strand cDNA for VSV. The high recovery of VSV using this approach combined with the genetic malleability of the VSV cDNA has useful applications to vaccine development. Gene expression of the non-segmented negative-strand RNA viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter (Wagner, 1996). The rearrangement of these genes appears to affect virus phenotype and although live attenuated viruses can be generated, they appear to have reduced capacity to generate clinical disease in the natural hosts such as the domestic swine (Flanagan et al., 2001). Nevertheless, these attenuated viruses were still effective delivery mechanisms for generating VSV-G immune responses and protection against wild type VSV following challenge of vaccinated animals (Flanagan et al., 2000; Flanagan et al., 2001).

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In subsequent studies, it was demonstrated that by introducing an extra foreign transcription unit between G and L proteins, rVSVs could be recovered that highly expressed and efficiently incorporated foreign proteins into their virions (McKenna et al., 2003; Schnell et al., 1996). These include CD4 receptor, measles virus glycoprotein (Schnell et al., 1996), and influenza virus hemagglutinin (HA) and neuraminidase (NA) (Kretzschmar et al., 1997). Furthermore, EM analysis of rVSV-HA/NA influenza particles demonstrated that the recombinants were mosaics carrying both VSV-G and influenza glycoproteins. There appears therefore significant space in the VSV membrane that can accommodate foreign membrane proteins.

From the features described above it is clear that vaccines based on live VSV recombinants have advantages over other live recombinant vaccine vectors. First, compared to large complex genomes of the Poxviridae family that encode numerous proteins that include immunoevasive and immunosuppressive proteins, the VSV genome is relatively simple, well understood and easier to manipulate (Lawson et al., 1995). Second, there is a potential for generating live attenuated viruses with reduced pathogenic phenotypes (Flanagan et al., 2000). Third, compared to segmented genomes of viruses in the Orthomyxoviridae family, the single-stranded genome of VSV does not undergo re-assortment and therefore these attenuated viruses cannot genetically recombine with wild-type viruses in vivo.

RECOMBINANT VSV VACCINE STUDIESVSV infection can be neuropathic in mice, following high dose intranasal infection, since olfactory receptors are highly tropic for VSV (Bi et al., 1995). Lethal encephalitis can occur especially in young mice but the virus can also be cleared by innate and adaptive immune responses (Balachandran et al., 2000a, Durbin et al., 1996; Meraz et al., 1996; Muller et al., 1994). Several recombinant VSV studies against human pathogens have been tested in murine models. For example, antibodies to influenza glycoprotein HA appear to be neutralizing in natural infection and a recombinant VSV-HA (rVSV-HA) has been shown to successfully generate protective immunity to lethal doses of influenza A/WSN/33 (H1N1) virus in BALB/c strain of mice (Roberts et al., 1998). Furthermore, the rVSV-HA was administered intranasally to elicit immune responses to the expressed influenza HA protein. As described above, the rVSV cloned from cDNA appears to have reduced pathogenicity compared to wild-type VSV Indiana strain. In related studies, an attenuated live rVSV expressing influenza HA with a truncation of the cytoplasmic domain of the VSV-G protein was intranasally administered to mice and found to completely protect against influenza challenge (Roberts et al., 1999). Similarly, in the same study a non-propagating rVSV devoid of VSV-G was also capable of eliciting protective immunity to influenza HA. This vector was also non-pathogenic and additional advantages over its replication counterpart include a

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lack of neutralizing antibody stimulation against the vector itself and the replication defective strategy improves potential safety attributes as a viral vaccine vector.

In the case of respiratory syncitial virus (RSV), replication competent or attenuated non-propagating VSV expressing RSV G (attachment) and RSV F (fusion) glycoproteins has been reported (Kahn et al., 2001). The VSV-RSV-F virus appeared to elicit RSV-specific antibodies in serum as well as neutralizing antibodies to RSV that were found to be protective against RSV challenge in BALB/c mice.

Finally, the rVSV approach has also been successfully applied to higher animal models. For example an AIDS vaccine based on attenuated VSV vectors expressing SHIV env and gag genes was tested in rhesus monkeys (Rose et al., 2001). This approach provided significant protection as demonstrated in vaccinated animals challenged with pathogenic AIDS virus. Since VSV was shown to be a potent inducer of cellular and humoral immunity in several viral models, we considered VSV as a tool for delivering HCV immunity.

VSV EXPRESSING HCV CORE, E1 AND E2 STRUCTURAL PROTEINS AS A POTENTIAL VACCINE AND IMMUNOTHERAPEUTIC STRATEGY AGAINST HCV INFECTIONIn our laboratory we have generated VSV-based HCV vaccine vectors that express all the HCV structural proteins, to maximize an immune response to multiple HCV epitopes (Ezelle et al., 2002; Majid et al., 2006). In the first strategy we developed a recombinant live or replication competent VSV antigen delivery system (Ezelle et al., 2002). To accomplish this, the contigous HCV core, E1 and E2aa 1-746 (HCV

Table 1. CTL activation by VSV-HCV-C/E1/E2 following intravenous injection. IFNγ ELISPOT analysis was determined by ELISPOT. Splenocytes were harvested from intravenously vaccinated BALB/c mice 4 weeks after initial injection and pulsed against core, E1, or E2 peptides shown. IFNγ production is illustrated in VSV-HCV-C/E1/E2 (VSV-C/E1/E2) mice (Ezelle et al., 2002).

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Fig. 1. Construction of rVSV (VSV-HCV-C/E1/E2) expressing HCV Core, E1 and E2. HCV NIHJ1 (genotype 1b provided by T. Miyamura) Core, E1 and E2 regions (aa 1-746) were cloned into the pVSV-XN2 vector (provided by J. Rose). Co-transfection of the recombinant pVSV-XN2-C/E1/E2 with vectors pBL-N, pBL-P, and pBL-L into BHK cells previously infected with vaccinia expressing T7 polymerase (v-TF7-3), results in transcription, translation and replication of VSV-HCV-C/E1/E2. The infectious rVSV virions are released from the cell (Ezelle et al., 2002, Lawson et al., 1995).

genotype 1b) open reading frame (ORF) was cloned into the Indiana (VSVI) cDNA backbone encoded from the plasmid pVSV-XN2 (Fig. 1). In order to create recombinant virus, BHK cells are infected with vaccinia encoding the T7 polymerase (vTF7-3) that drives expression of the N, P and L proteins of VSV encoded in pBL-N, pBL-P, and PBL-L plasmids respectively. Co-transfection of the attenuated VSV cDNA genome vector (pVSV-XN2-C/E1/E2) in this set up allows for efficient recovery of recombinant VSV (rVSV) expressing foreign genes (as described above).

The methodology above allows for large scale preparations of replication competent rVSV for cell and animal studies. Importantly, we observed only slight attenuation in the growth properties of replication competent VSV-HCV-C/E1/E2 as compared to wild type virus counterparts as shown in Fig. 2 (Ezelle et al., 2002). Furthermore,

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VSV-HCV-C/E1/E2 expressed high levels of HCV antigen illustrated in Fig. 3 (Ezelle et al., 2002). In recent studies, analysis using a panel of conformational sensitive mouse and human antibodies illustrated that rVSV expressed authentically folded non-covalently linked E1/E2 heterodimers (Ezelle et al., 2002; Majid et al., 2006).

Fig. 2. Growth curve analysis of rVSV. VSV-HCV-C/E1/E2 demonstrates a similar growth rate to VSV-GFP. Infections were undertaken at an MOI of 1 for 30 min. Cell medium was analyzed for viral titers at 6, 12, 18, and 24hr post-infection by standard plaque assay (Balachandran et al., 2000b, Ezelle et al., 2002).

Fig. 3. Expression of HCV core, E1, and E2. BHK cells were infected with VSV-HCV-C/E1/E2 (VSV-C/E1/E2) or control wild type VSV (VSV-XN2) at an MOI of 1. Cell lysates were analyzed for HCV protein expression by immunoblot analysis 18 hr post-infection. The results demonstrate that VSV can be used to express high levels of HCV structural antigens (Ezelle et al., 2002).

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The binding of these VSV expressed HCV glycoproteins to patient antibodies from native HCV infection is an important potential recognition of these immunogens in HCV vaccine design. Furthermore, intravenous or intraperitoneal immunization of BALB/c mice with VSV-HCV-C/E1/E2 elicited potent secondary serum antibody responses to HCV E2 as detected by ELISA demonstrated in Fig. 4 (Ezelle et al., 2002). Vaccine regimens involved intravenous injections with VSV-HCV-C/E1/E2 or control VSV-GFP, or PBS followed by a secondary inoculation 2 weeks later. The sera were tested for antibody responses on day 21 post initial injection. Our data indicated that VSV-HCV-C/E1/E2 is an efficient vehicle for generating HCV E2 antibodies and warrant further study to assess their capacity in neutralizing HCV infection. In addition, we have observed that VSV-HCV-C/E1/E2-injected mice produced strong HCV core antibodies indicating that immune responses to all the HCV structural proteins were being successfully generated.

Fig. 4. Humoral immunity generated to HCV structural antigens using VSV. (A) Sera were collected from BALB/c mice vaccinated by intravenous injection (6 mice per group) with VSV-HCV-C/E1/E2 (VSV-C/E1/E2), VSV-GFP or PBS only. Anti-HCV-E2 was detected only in VSV-HCV-C/E1/E2 group when analyzed in an ELISA format against recombinant E2 protein (Ezelle et al., 2002). (B) Intraperitoneal administration of VSV-C/E1/E2 can also generate humoral immunity to HCV E2 in BALB/c animals.

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In addition to strong humoral responses, the generation of multispecific CTL responses are proposed to be essential for clearance of HCV during acute infections in humans and chimpanzees (Cooper et al., 1999; Erickson et al., 2001; Neumann-Haefelin et al., 2005; Thimme et al., 2001). Therefore, to determine if VSV-HCV-C/E1/E2 was able to generate CD8+ T cell responses, we investigated CTL responses in splenocytes from vaccinated mice using IFN-γ ELISPOT analysis against HCV genotype 1b peptides known to stimulate CTL activity in BALB/c models (Gordon et al., 2000; Nishimura et al., 1999). The release of IFN-γ cytokine from CD8+ T cells is an indication of Th1 type immunity, and our studies illustrated that VSV-HCV-C/E1/E2 could indeed elicit Th1 type CTL responses against HCV core, E1 and E2 peptides 4 weeks after the initial injection as illustrated in Table 1 (Ezelle et al., 2002).

We have also evaluated whether routes of inoculation using rVSV could affect the strength and type of immunity generated against HCV antigens. Intraperitoneal injections were performed using VSV-HCV-C/E1/E2, VSV-GFP or PBS. Serum collected on day 28 was tested again in an ELISA format and demonstrated significant anti-E2 antibody levels in VSV-HCV-C/E1/E2 injected mice shown in Fig. 4B (Ezelle et al., 2002). Interestingly, splenocytes harvested 7 days after injection demonstrated CTL responses to HCV structural peptides as shown by IFN-γ ELISPOT and presented in Table 2 (Ezelle et al., 2002). Collectively, our data indicate that rVSV expressing HCV antigens can stimulate potent humoral and cellular immunity in animal models. Studies ongoing in our laboratory also suggest that a non-propagating VSV strategy may also be a feasible option for generating immunotheraputic intervention to combat HCV infection. Given the genetic malleability of VSV, the possibility of generating a number of vectors that

Table 2. CTL activation by VSV-HCV-C/E1/E2 following intraperitoneal route of inoculation. Splenocytes were harvested and analyzed for IFNγ production by ELISPOT 7 days following the injection. The results indicate again that only VSV-HCV-C/E1/E2 (VSV-C/E1/E2) vaccinated mice activate T-cells when pulsed with HCV peptides (Ezelle et al., 2002).

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are safe and yet very effective at generating immune responses to HCV remain an exciting prospect.

CONCLUSIONSThe window against the quest for developing HCV vaccines and immunotherapy has certainly shortened as result of a wealth of knowledge distributed with regard to the immune responses that appear to be hallmarks of acute HCV clearance in chimpanzees and humans. In addition, understanding key targets for virus neutralization, particularly the HCV glycoprotein complex will aid the design of immunogens and generate effective immune responses potentially to epitopes broadly conserved amongst viral types. Furthermore, it appears increasingly likely that viral neutralization by antibody alone may not be sufficient in generating effective immune responses against a prophylactic HCV vaccine approach. Nevertheless, antibody responses alone using subunit vaccines appears to reduce chronicity of HCV infection, suggesting a therapeutic role in preventing HCV related liver disease that may account to decreased morbidity and mortality in patients. Indeed, an HCV glycoprotein-based subunit vaccine trial in humans demonstrated a potential immuno-therapeutic role for vaccination (Nevens et al., 2003).

The innovation of new technologies that can elicit potent humoral and cellular responses to multi-specific HCV antigens is likely key to a first line of defense against HCV infection. Several promising approaches have been described in this work. However, many studies are in preliminary phases using small animal models and their findings require confirmation in HCV animal models, such as the chimpanzees. The ability to effectively 'tune' individual immune responses against key HCV antigens; core, E1, E2 (and NS3) may provide an opportunity for the immune system to prevent HCV infection before this virus can outpace the immune system as described in chronically infected individuals (Willberg et al., 2003). In this regard, the VSV system described in this report offers significant promise since this recombinant viral approach appears to be a potent stimulator of HCV immunity in the murine model. Furthermore, several features of this system, for example, the malleability of the VSV genome, ability to recovery high titer replication competent or non-propagating recombinant viruses, high level expression of foreign genes, and lack of pathogenicity in humans makes VSV an exciting tool for further endeavors in development of a vaccine against HCV infection.

FUTURE TRENDSAlthough there are still technical and immunological obstacles to overcome, many exciting technologies being tested may improve vaccine studies and also immunotherapy. Essential antigen presenting cells that initiate the immunological cascade, such as dendritic cells (DCs) have been proposed to be impaired during HCV infection although initial reports have been controversial and recent studies

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suggest normal functions of circulating plamacytoid or myeloid DCs (Bain et al., 2001; Longman et al., 2005; Sarobe et al., 2003). In any case, it may be possible to overcome this defect by autologous transfusion of HCV antigen-loaded mature DC (Gowans et al., 2004). In addition, self replicating cytopathic and non-cytopathic replicon transfection of DCs ex-vivo, illustrates efficient processing of HCV antigens and stimulates efficient priming of T-cell responses following transfusion into murine models (Racanelli et al., 2004). Furthermore, HCV-VLP uptake and presentation has also been demonstrated by human DC (Barth et al., 2005). Key goals therefore include better antigen delivery platforms and priming of efficient immune responses.

The non-propagating VSV approach offers enhanced safety for a recombinant viral vector. This virus can infect many cells, including DC, and can effectively activate unknown innate responses that will inevitably facilitate efficient transduction of adaptive immunity (Balachandran et al., 2000a, Balachandran et al., 2004). Furthermore, modification of these viruses to express cell specific markers for targeting or cytokines for adjuvant purposes is plausible. Finally, discovery of novel innate intracellular molecules that are involved in anti-viral host defense may also be inserted into VSV-based vectors to further facilitate potency of vaccine against virus infection (Balachandran et al., 2004). These exciting advancements and better understanding of HCV immunology suggest an adventurous future in the quest for prophylactic and or immunotherapy against HCV infection.

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Infectious HCV Systems

Chapter 16

Development of an Infectious HCV Cell Culture System

Takaji Wakita and Takanobu Kato

ABSTRACTHepatitis C virus (HCV) infection causes chronic liver diseases and is a health problem worldwide. Despite the increasing demand for knowledge on viral replication and pathogenesis, detailed examinations of the viral life cycle have been hampered by the lack of efficient viral culture systems, owing in part to its narrow host range. We isolated full-length HCV clone, JFH-1strain, from a fulminant hepatitis C patient. The JFH-1strain fit into the cluster of genotype 2a with notable deviations in the 5'-untranslated region (5'UTR), core, NS3 and NS5A regions, and monoclonality of the hyper-variable region sequence. The JFH-1 subgenomic replicon replicated efficiently in a variety of cell lines without acquiring adaptive mutations in its genome. Transfection of in vitro transcribed full-length RNA into Huh7 cells, efficient replication of JFH-1 RNA and secretion of recombinant viral particles into culture medium. Importantly, secreted viral particles were infectious for both cultured cells and a chimpanzee. Furthermore, infectivity for cultured cells was improved by using permissive cell lines. This infectious HCV system provides for the first time a powerful tool to study the full viral life cycle, to construct anti-viral strategies and to develop effective vaccines.

INTRODUCTIONEfforts to understand the viral life cycle of hepatitis C virus (HCV) and to identify effective antiviral agents have been hampered by the lack of an efficient cell culture system for this virus. Many attempts to develop a system for HCV infection and replication in cell culture have already been undertaken; in fact, some advances have been reported (Bertolini et al., 1993; Ito et al., 1996; Mizutani et al., 1996; Iacovacci et al., 1997; Fournier et al., 1998; Rumin et al., 1999; Ito et al., 2001; Zhao et al., 2002; Zhu et al., 2003). However, the viral replication efficiencies reported in these studies were modest, requiring detection by a reverse transcription polymerase chain reaction (RT-PCR). We hypothesized that the replication ability of HCV may differ among HCV clones. We therefore isolated an HCV clone, JFH-1, from a fulminant hepatitis patient with HCV (Kato et al., 2001). JFH-1-derived subgenomic replicon proved capable of higher replicative capacity in a variety of cell lines, and production of infectious HCV particles in Huh7 cells.

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A CASE OF FULMINANT HEPATITIS ASSOCIATED WITH HCVIn 1999, we obtained sera from a fulminant hepatitis patient (Kato et al., 2001). The 32-year-old male patient was admitted with general fatigue, high-grade fever, and liver dysfunction. No evidence of prior liver disease was found, and the patient had no history of drug or alcohol consumption. In the previous 6 months, he had not received any blood transfusions, taken any drugs intravenously, undergone acupuncture, nor had sexual contact with a known hepatitis virus carrier. This patient showed high levels of serum aspartate aminotransferase and alanine aminotransferase, low levels of the minimum prothrombin time value, and displayed stage II encephalopathy. HCV RNA was detected by RT-PCR, and anti-HCV antibody was negative. All other hepatitis viral markers, anti-HAV antibodies (IgG and IgM), hepatitis B virus (HBV) markers (HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc and HBV-DNA), and GB virus-C RNA, were negative. Therefore, he was diagnosed as having HCV-associated fulminant hepatitis. The infection route of HCV was obscure. The patient showed high levels of viremia, 105 copies/ml at admission and 104 copies/ml 25 days later. However, HCV was undetectable at 65 days after admission, at which point anti-HCV antibody was positive. At 75 days after admission, his condition improved and he was discharged from the hospital.

To investigate the role of strain-specific viral characteristics of HCV in fulminant hepatitis, we isolated HCV RNA from the acute phase serum of this patient, amplified it by RT-PCR, and determined the sequence of its entire genome.

SEQUENCE ANALYSIS OF JFH-1The HCV clone isolated from the fulminant hepatitis patient, designated JFH-1, was determined to be of genotype 2a. To compare genomic characteristics, we also determined the entire genomic sequences of 6 HCV genotype 2a clones isolated from 6 chronic hepatitis patients (JCH-1 to -6). JFH-1 is 9,678 nucleotides (nt) in length with a long open reading frame spanning nt 341-9439 coding for 3033 amino acids (aa). Clones isolated from 6 chronic hepatitis patients, JCH-1 to -6, comprised 9681, 9677, 9678, 9676, 9691, and 9686 nt, respectively, and encoded either 3032 or 3033 aa. In phylogenetic analysis, JFH-1 clustered with other genotype 2a clones, but showed slight deviation from clones isolated from chronic hepatitis patients, JCH-1 to -6, and HC-J6 (prototype of HCV genotype 2a, accession number is D00944) (Fig. 1). To determine the degree of deviation in each subgenomic region or entire genome, the ratios of mean genetic distances [ratio = mean genetic distance between JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) in each subgenomic region or entire genome / mean genetic distance among all 2a strains in each subgenomic region or entire genome] were calculated. For nucleotide analysis of the entire genomes, the mean genetic distances between JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) and among all 2a strains were calculated to be 0.1136±0.0073 and

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0.0969±0.0140, respectively, with the ratio of mean genetic distances representing the deviation of clone JFH-1 among genotype 2a clones being 1.173 (Table 1). Among analyses of each subgenomic region, the 5'UTR showed the greatest ratio of mean genetic distances, 1.387, and was identified as the region with the greatest deviation. For amino acids, mean genetic distances of the entire genome between JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) and among all 2a strains were 0.0918±0.0052 and 0.0716±0.0139, respectively, giving a ratio of mean genetic distances of 1.282. Analyses of each the subgenomic region revealed greater diversity for core, nonstructural (NS) 3, and NS5A, with mean genetic distance ratios of 1.560, 1.464, and 1.596, respectively. The complexity of HCV infection in the fulminant hepatitis patient was also assessed by determining the distribution of quasispecies in the hyper-variable region (HVR). Sequences of the 20 amplified clones of the envelope (E) 2 region were determined and the frequencies of these sequences were examined at the two time points at days 1 and 23 after admission. In the early point of acute phase (day 1 after admission), 17/20 HVR sequences were identical and those of the other 3 clones showed a difference of only one aa substitution. At the later time point of the acute phase (day 23 after admission), the HCV clones were identical (20/20,). These data suggest that the HCV in this patient showed lower complexity than that of the general viral population. Monoclonality of the viral population has also been reported for another case of HCV related

Fig. 1. Phylogenetic tree based on the entire HCV genome of for JFH-1, JCH-1 to -6 and representative strains for which the entire genome has been reported. The number of nucleotide substitutions per site at each position was estimated by the six-parameter method, and a phylogenetic tree was drawn using the neighbor-joining method. The length of the horizontal bars indicates the number of nucleotide substitutions per site.

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fulminant hepatitis (Farci et al., 1996). Thus, we speculated that monoclonality of the viral population is related to the development of fulminant hepatitis and that the JFH-1 clone, especially in the 5' UTR, core, NS3 and NS5A regions, has some specific viral characteristics related to fulminant hepatitis.

PREFERENTIAL PROCESSING FOR CORE PROTEIN OF JFH-1Among the subgenomic regions of JFH-1, 5'UTR, core, NS3 and NS5A were identified as deviated regions. Among these regions, core protein is known to form the viral particle and also to regulate multiple functions in host cells (Moriya et al., 1998; Ray et al., 2001; Watashi et al., 2003; see Chapter 3). During virus assembly, the core protein undergoes two consecutive membrane-dependent cleavages, and it develops into two forms, p23 and p21 (Liu et al., 1997). The p21 core protein is cleaved from the endoplasmic reticulum-bound p23 core protein or the longer precursor polyprotein by host signal peptide peptidase (McLauchlan et al., 2002). The p21 core protein was predominantly observed in patient serum containing native viral particles (Yasui et al., 1998). Thus, the p21 core protein is the mature and stable form that accumulates in the cell and eventually constitutes the viral capsid. We investigated the differences in p21 core protein production between JFH-1 and the other genotype 2a clones isolated from chronic hepatitis patients (JCH-1 to -5) (Kato et al., 2003a). Using the core or core-E1 expression vector of JFH-1 and JCH-1, we found that JFH-1 could preferentially produce the p21 core protein in

Table 1. Ratios of mean genetic distance for each subgenomic region.Region Nucleotide Amino Acid5'UTR 1.387 NA**Core 1.251 1.560E1 0.986 0.940E2 1.107 1.066NS2 1.243 1.298NS3 1.168 1.464NS4A 1.249 1.044NS4B 1.178 1.223NS5A 1.222 1.596NS5B 1.213 1.2083'UTR 0.989 NAEntire Genome 1.173 1.282*Ratios were calculated using the mean genetic distances [ratio = mean genetic distance between JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) in each subgenomic region or entire genome / mean genetic distance among all 2a strains in each subgenomic region or entire genome] (Kato et al., 2001).**NA, not available

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both in vitro translation assay and cell transfection assay with Huh7, HepG2 and HeLa cells. Similar results were also obtained when comparing JFH-1 with the other clones (JCH-2 to -5) isolated from chronic hepatitis patients. Investigations with chimeric constructs revealed that differences in core protein processing depend on the c-terminal region of the core protein. We identified 4 aa substitutions in this region of the core protein between JFH-1 and the other clones isolated from chronic hepatitis patients. Through experiments with mutation-introduced constructs, all 4 of these aa of JFH-1 were found to be responsible for the preferential production of p21 core protein. Based on these findings, we suspected that JFH-1 may be able to preferentially produce viral particles over other HCV clones.

REPLICATION CAPACITY OF JFH-1 AS A SUBGENOMIC REPLICONTo investigate the function of the NS region of JFH-1, we constructed a subgenomic replicon system using this clone. The HCV subgenomic replicon system has enabled us to mimic HCV replication in Huh7 cells, and has been used as a tool in the study of the mechanism of HCV replication (Lohmann et al., 1999; see Chapter 11). JFH-1 showed higher colony formation efficiency that was approximately 500-fold more efficient than the prototype Con-1 replicon and 50-fold more efficient than the Con-1/NK5.1 replicon, which contains highly adaptive mutations (Kato et al., 2003b). Furthermore, the JFH-1 replicon could replicate efficiently not only in Huh7 cells, but also in other hepatocyte-derived cell lines, HepG2 and IMY-N9 cells (Date et al., 2004), and non- hepatocyte derived cell lines, HeLa and 293 cells (Kato et al., 2005). This result may be attributed to the replication proficiency of JFH-1. Importantly, the JFH-1 replicon did not require an adaptive mutation in order to replicate in these cell lines. Most clones isolated from each of these cell lines showed no or a few aa mutations in the HCV-derived replicon regions. Previously, Bukh et al. (2002) demonstrated that HCV infection could not be achieved with full-length HCV RNA containing multiple cell-culture adaptive mutations. Thus, the higher replication capacity and the absence of adaptive mutations of JFH-1 may be important for developing an infectious HCV system.

CONSTRUCTION OF FULL-LENGTH JFH-1 cDNABased on results obtained using subgenomic replicons, we found that the JFH-1 strain replicates very efficiently in Huh7 cells, as shown not only by colony formation assay with G418 selection, but also by transient replication assay (Kato et al., 2003a; 2003b; Date et al., 2004; Kato et al., 2005). This suggests that the JFH-1 genome can replicate autonomously in Huh7 cells without the help of G418 selection pressure and the development of adaptive mutations. Taking advantage of the efficiency of the JFH-1 strain replication capacity, we planned to test the replication of a full-length JFH-1 clone in Huh7 cells.

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Monoclonality is one of the specific characteristics of HCV strains in fulminant hepatitis, and the JFH-1 strain, as confirmed by isolations made from other patients (Farci et al., 1996; Kato et al., 2001). This characteristic is also advantageous in the construction of consensus clones in the production of full-length cDNA because, usually, HCV possess a wide variety of mutations called quasispecies (Martell et al., 1992). Thus, it was necessary to inject 10 different clonal mixtures into a chimpanzee to establish the first infectious clone for chimpanzee (Kolykhalov et al., 1997). On the other hand, JFH-1 cDNA was cloned from RT-PCR fragments and, although some sequence diversity was present, the aa sequences were highly conserved and full-length HCV cDNA encoding the JFH-1 strain consensus sequence was easily assembled by connecting the cloned PCR fragments (Kato et al., 2001; Wakita et al., 2005). The T7 promoter sequence was inserted just upstream of the full-length JFH-1 cDNA sequence, and full-length synthetic JFH-1 RNA was transcribed from pJFH-1 by T7 RNA polymerase.

REPLICATION OF FULL-LENGTH JFH-1 RNA IN Huh7 CELLSWe first transfected in vitro transcribed full-length JFH-1 RNA into naïve Huh7 cells, which is the original cell line used for subgenomic replicon studies. As we expected, full-length JFH-1 RNA replicated efficiently in the transfected cells, as determined by Northern blot analysis (Wakita et al., 2005). Viral proteins produced from replicated RNA were demonstrated by immunofluorescence and Western blot analyses. Transfection of replication incompetent mutant RNA transcribed from pJFH1/GND, in which GDD catalytic motif of NS5B was mutated to GND, into Huh7 cells, however, did not lead to viral replication or protein production.

We expected to achieve replication of full-length RNA in transfected Huh7 cells because full-length genotype1b RNA with adaptive mutation had been reported to replicate in Huh7 cells and subgenomic replicons of the JFH-1 strain had been shown to produce more colonies in Huh7 cells than genotype1b replicons (Ikeda et al., 2002; Pietschmann et al., 2002; Kato et al., 2003b). However, it was difficult to predict viral particle formation and secretion because these had not been achieved by full-length HCV RNA transfection, even though RNA replication was observed in the transfected Huh7 cells (Ikeda et al., 2002; Pietschmann et al., 2002). To determine whether the viral particles were formed and secreted into the culture medium from the full-length JFH-1 RNA transfected cells, we performed several biological assays. First, we analyzed the density of secreted viral proteins and viral RNA by sucrose density gradient. It has been reported that the supernatant of full-length replicon RNA replicating cells of the Con1 strain secrete viral RNA into culture medium; however, the density of viral RNA was found to have a very similar culture medium density as that from subgenomic RNA replicating cells (Pietschmann et al., 2002). We thus first analyzed an aliquot of culture supernatant from full-length JFH-1 RNA transfected cells by sucrose density gradient. Following

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ultracentrifugation, 16 fractions were obtained from the bottom of the tube. Both viral core protein and RNA were quantified using sensitive core ELISA (Aoyagi et al., 1999) and RT-PCR with real-time detection, respectively (Takeuchi et al., 1999). Interestingly, both core protein and RNA peaks occurred in the same fraction (around 1.17 g/ml), a density greater than the one where subgenomic replicon cells usually segregate. Next, we determined RNase sensitivity of these peaks, as the viral RNA genome packed in the particles should be protected from RNase digestion in culture. Culture medium from the transfected cells were RNase digested, followed by density centrifugation. The profile analysis of the density peaks revealed that RNase digestion did not change the density gradient distribution of both RNA and core protein, indicating the viral genome was protected from nuclease digestion (Wakita et al., 2005).

Next, we confirmed whether the envelope proteins were incorporated into secreted viral particles. If the viral particles are properly and completely formed and secreted, viral genome and core protein form a nucleocapsid and are surrounded by envelope proteins (E1 and E2 proteins). To assay envelope proteins, we treated culture medium with detergent to strip the envelope components from the viral particles. Viral envelope usually comprises cellular membrane components such as lipids, making the density of envelope lighter than that of the inner nucleocapsids. We found that both core protein and RNA peak fractions became heavier (around 1.25 g/ml), indicating the removal of the lighter envelope components by the detergent treatment. Furthermore, we demonstrated the incorporation of both E1 and E2 proteins by Western blot analysis of peak fractions of viral RNA after density gradient centrifugation. We collected approximately 2.5 litter of culture medium from transfected cell cultures. Culture medium was concentrated by ultrafiltration and then by ultracentrifugation, and was then fractionated by sucrose density gradient. Each collected fraction (counted from the bottom of the centrifuge tube) was further concentrated by ultrafiltration. Concentrated fractions were separated by SDS-PAGE and then transferred onto PVDF membrane. Core, E1 and E2 proteins were detected on each fraction using specific antibodies. Thus, all the components of the viral particle were detected in the same density gradient fraction, suggesting proper viral particle formation and secretion. Finally, viral particles secreted into the culture medium were visualized by immuno-electron microscope analysis using anti-E2 monoclonal antibody. Viral particles were shown to be spherical, with an outer diameter of about 55 nm (Wakita et al., 2005).

INFECTIVITY OF SECRETED VIRAL PARTICLES FROM JFH-1 TRANSFECTED CELLSAfter having confirmed the presence of secreted viral particles, we were interested in the infectivity level of secreted viral particles. We used double-chambered culture plates equipped with polyester membrane (0.45 μm pore size) separating the inner

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and outer chambers. Thus, substances smaller than this pore size, such as virus particles, can diffuse across the membrane and populate both chambers. Full-length JFH-1 RNA transfected cells were transferred to the inner chamber and naïve Huh7 cells were seeded in the outer chamber. A few days after the start of the experiment, naïve Huh7 cells in the outer chamber were stained with anti-HCV antibodies to confi rm infection by secreted virus particles. To our surprise, a few cells were positively stained, although at very low frequency. To confi rm that infection occurred for naïve Huh7 cells, we collected culture medium of transfected Huh7 cells, which was subsequently cleared by low speed centrifugation and fi ltered through a disk fi lter (0.45 μm-pore size). Naïve Huh7 cells were inoculated with the cleared free virus in a culture plate for 3 hours. Inoculated cells were then washed with PBS and cultured for another 48 h in complete medium. To increase infection effi ciency, culture medium was concentrated by ultrafi ltration. Inoculation of concentrated culture medium increased the numbers of infected cells, however, the effi ciency was still low at around 0.5% with Huh7 cells. Inoculated cells were harvested after infection, and HCV RNA titer was determined by PCR with real time detection (Fig. 2a). Only 1% of inoculated HCV RNA was adsorbed by inoculated cells and HCV RNA copies in the infected cells were further decreased within 12 hours after inoculation. However, RNA titer in the infected cells increased at 24 hours after inoculation. Core protein expression measured in infected cells by sensitive ELISA showed a decrease within 12 hours after inoculation and an increase at 24 hours after infection (Fig. 2b). These data clearly showed that viral particles were infectious for Huh7 cells, although at low effi ciency (Wakita et al., 2005).

Fig. 2. HCV RNA replication (a) and core protein production (b) in infected Huh7 cells. Culture medium was collected from full-length JFH1 RNA-transfected cells and concentrated by ultrafi ltration. Naïve Huh7 were seeded 24 h before infection. Filtered (0.45-µm pore size) culture media was inoculated for 2 h with periodic rocking. After inoculation, cells were washed with PBS and were cultured in complete culture medium for another 12, 24, 48, 72, and 96 h. Experiments were performed in triplicate, and mean titers (closed circles) SD (bars) are shown.

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NEUTRALIZATION OF JFH-1 INFECTIVITY BY CD81 ANTIBODY AND PATIENT SERACD81 has been identified as an E2 protein binding protein (Pileri et al., 1998). We thus tested the effectiveness of anti-CD81 antibody for inhibiting infectivity of culture supernatant for naïve Huh7 cells. Naïve Huh7 cells were treated with 10 μg/ml of anti-CD81 antibody at room temperature and then washed with PBS followed by inoculation with culture medium from transfected cells (Wakita et al. 2005). HCV RNA titer in the inoculated cells was inhibited more than 1 log, indicating that infection by secreted viral particles is at least partially dependent on a CD81-specific pathway. Further studies will be necessary to determine whether CD81 is a sole receptor molecule involved in adsorption and internalization steps or whether other molecules are also involved.

The neutralizing activity in chronically infected patient sera has been shown by experiments using pseudotype virus harboring HCV envelope proteins (Bartosch et al., 2003; Yu et al., 2005; Logvinoff et al., 2004). We also tested some patient sera for neutralizing activity against JFH-1. To increase sensitivity of the assay, a bicistronic replicon construct containing luciferase reporter was used (Wakita et al. 2005). Indeed, patient serum tested positive for neutralization of virus infection proved to contain some neutralizing antibodies against JFH-1 (Wakita et al., 2005).

IN VIVO INFECTIVITY OF JFH-1 CULTURE MEDIUMTo further confirm authenticity of the viral particles produced in our study, in vivo infectivity was tested in a chimpanzee (Wakita et al., 2005). Electroporated culture supernatant was harvested from the full-length JFH-1 RNA-transfected cells and cleared by low-speed centrifugation and then passed through a 0.45-μm disk filter. Control culture medium was prepared from the cells mixed with JFH-1 RNA, but with the omission of electroporation pulse. A chimpanzee was first inoculated with undiluted control culture medium, and no infection was observed. Then, 104 diluted culture medium harvested from the transfected cell was used for inoculation, but again, no infection developed. Six weeks later, 103 diluted culture medium was inoculated in the same subject, and viremia was induced. Viral titer was low, with the highest HCV RNA titer being 2.04x103 copies/ml. Furthermore, HCV infection was cleared without any evidence of abnormal liver histology or elevation of liver-specific enzymes or HCV-specific antibody seroconversions (Wakita et al., 2005). Further investigation is necessary to determine whether the nonvirulent phenotype is a characteristic of the JFH-1 strain.

PERMISSIVE CELLS FOR JFH-1 INFECTIONThe infection efficiency of JFH-1 was quite limited as only a small percentage of the inoculated cells appeared positive for HCV by antigen staining (Wakita et al., 2005). To increase the infection efficiency, specific cell lines derived from

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Huh7 cells were analyzed by several groups. Huh7.5, which is one of the cured cell lines established from original HCV replicon cell lines, supported high levels of subgenomic HCV replication with Con1 and H77 strains (Blight et al., 2002). Huh7.5.1 is a cell line derived from Huh7.5 (Zhong et al., 2005). Indeed, infectivity of Huh7.5 or Huh7.5.1 cell line with JFH-1 was markedly increased (almost 100%) compared to standard Huh7 cells (Lindenbach et al., 2005; Zhong et al., 2005). Furthermore, Zhong and colleagues (2005) also were able to prevent in vitro-produced virus from infecting Huh7.5.1 using an anti-CD81 antibody, whereas Lindenbach and his coworkers (2005) accomplished this with Huh7.5 by using a soluble recombinant CD81 fragment.

SUMMARY AND CONCLUDING REMARKSRecombinant HCV particles were produced and secreted from JFH-1 RNA-replicating cells, and the secreted viruses were infectious to both Huh7 cells and a chimpanzee (Zhong et al., 2005; Lindenbach et al., 2005; Wakita et al., 2005). Biophysical property analysis showed that cell culture-grown virus particles have a density of about 1.15 – 1.17 g/ml, are spherical, and have an outer diameter of about 55 nm (Wakita et al., 2005). Both the density and the overall diameter of the particle are in agreement with a recent report describing the production of virus particles with a DNA-based expression system (Heller et al., 2005). Infectivity can be significantly neutralized by CD81-specific antibodies, supporting observations that CD81 plays an important role in HCV cell entry made in HCV pseudo particles (Zhong et al., 2005; Lindenbach et al., 2005; Wakita et al., 2005; Bartosh et al., 2003; Hsu et al., 2003). Some level of neutralization was achieved with immunoglobulins in patient serum, showing that potentially protective antibodies are generated during chronic infection but that their capacity to prevent chronicity may be limited. We also observed cross-neutralization in sera from patients infected with a genotype 1 virus (Wakita et al., 2005).

Thus, JFH-1 is the first HCV strain with the capability to produce infection in tissue culture, and serves as a platform for a new generation of HCV investigations. Furthermore, the use of permissive cell lines such as Huh7.5 and Huh7.5.1 cell lines will further expedite full virus culture experiments in the laboratory. This infectious HCV system should provide opportunities to study the full HCV life cycle, including virus entry, replication, virus particle formation, and virus secretion, as well as to develop effective antivirals and vaccines.

ACKNOWLEDGEMENTSAnalysis of immuno-electron microscope and neutralization of infectivity of JFH-1 virus were done by Dr. Ralf Bartenschlager's group (University of Heidelberg, Heidelberg, Germany). In vivo experiment using a chimpanzee was done by Dr. T. Jake Liang's group (National Institute of Health, Bethesda, Maryland). Infection

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experiment using Huh7.5.1 cells was done by Dr. Frank Chisari's group (Scripps Research Institute, La Jolla, California). Supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Health, Labor, and Welfare of Japan, by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and by the Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.

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