identiﬁcation of residues in the hepatitis c virus core

JOURNAL OF VIROLOGY, June 2005, p. 6814–6826 Vol. 79, No. 110022-538X/05/$08.00�0 doi:10.1128/JVI.79.11.6814–6826.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Residues in the Hepatitis C Virus Core Protein ThatAre Critical for Capsid Assembly in a Cell-Free System

Kevin C. Klein,1 Sheri R. Dellos,1 and Jaisri R. Lingappa1,2*Department of Pathobiology, University of Washington,1 and Department of Medicine, University of Washington,2

Seattle, Washington 98195

Received 27 June 2004/Accepted 25 January 2005

Significant advances have been made in understanding hepatitis C virus (HCV) replication through devel-opment of replicon systems. However, neither replicon systems nor standard cell culture systems supportsignificant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently,we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cellextracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutationalanalysis to identify residues in HCV core that are important for capsid assembly in this highly reproduciblecell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids ofHCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, theaspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to besites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation ofprolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in thecell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These dataconstitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCVcore in capsid formation.

Hepatitis C virus (HCV) is a major public health concernworldwide. Two percent of the world’s population is infectedwith the virus (54), which is the major cause of non-A, non-Bhepatitis and which often leads to cirrhosis of the liver orhepatocellular carcinoma (42). While current treatments haveimproved, they are poorly tolerated, not completely efficacious,and not available in all settings (31). Development of bettertreatments and vaccines has been hampered by the lack ofvirus replication systems. Standard cell culture systems do notsupport HCV replication, and the chimpanzee is the only an-imal model capable of being infected and yielding high HCVtiters (23). These limitations have been partly overcome by thedevelopment of HCV replicon systems, which support auton-omous replication of HCV RNA (2, 10, 36, 37). Repliconsystems have allowed specific requirements of HCV RNA rep-lication to be studied; however, HCV particles, or even nu-cleocapsids, are not produced in these systems (9, 27, 53).Consistent with this observation, infectious particles are notreleased from these cells (27). As a result, the requirements forcritical steps in virion formation, including capsid assembly,genome encapsidation, budding, and release, remain largelyunknown.

HCV is an enveloped, single-stranded, positive-sense RNAvirus in the Flaviviridae family (3). The HCV genome has asingle open reading frame that codes for a �3,000-amino-acidpolyprotein. The structural proteins, core, E1, and E2, are theN-terminal products in the polyprotein and are released fromthe polyprotein by host proteinases (34, 43, 57). Once cleaved

from the polyprotein, the 173-amino-acid mature core proteinassembles into HCV capsids at the cytoplasmic face of theendoplasmic reticulum (ER) (7, 8, 45). Core is known to in-teract with the HCV envelope glycoprotein E1 at the ER (35),and assembled capsids are thought to acquire their envelopesby budding into the ER (4, 7, 8, 35). However, the specificdetails of these late events in the viral life cycle have not beenelucidated.

Besides forming the capsid that houses the HCV genomicRNA, core is also known to modulate diverse cellular func-tions. Core is carcinogenic when expressed in transgenic mice(47), has pro- and antiapoptotic functions (29), alters the tran-scription of other viral promoters (58, 59), and appears toinduce steatosis (48) and the formation of lipid droplets (1).Core is known to interact directly with many different cellularproteins that most likely play a role in modulating these diversefunctions (42) or may modulate the ability of core to assemble.Most interacting proteins have been identified through yeasttwo-hybrid approaches. All reported interactions of core withintracellular proteins, including the cytoplasmic domain of tu-mor necrosis factor (TNF) receptor 1 (65), lymphotoxin �receptor (15, 41), hnRNP K (26), DDX3 (51), Cap-Rf (64),and PA28� (46), are mediated by the N-terminal two-thirds ofcore. The observation that no cellular proteins associate withthe C terminus of core may reflect an artifact of the yeasttwo-hybrid assay (many screens have been performed withtruncations of core, e.g., 41) or may be because the hydropho-bic C terminus is less accessible to interactions with cellularproteins. However, the interaction of the HCV envelope gly-coprotein E1 with core has been mapped to the C terminus(35, 39). Core is also known to target to lipid droplets via its Cterminus, where it colocalizes with apolipoprotein AII (1).Deleting amino acids 153 to 169 or mutating the prolines at

* Corresponding author. Mailing address: Department of Pathobi-ology, Box 357238, University of Washington, 1959 NE Pacific St.,Seattle, WA 98195. Phone: (206) 616-9305. Fax: (206) 543-3873. E-mail: [email protected].

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positions 138 and 143 abolishes lipid droplet association (25,43). While the significance of core trafficking to lipid dropletsremains unclear, this targeting event may have implications forreplication or pathogenesis (42). Besides interacting with cel-lular proteins, core is known to self-associate. Amino acidsfrom 82 to 102, referred to as a homotypic interacting domain,mediate core-core interactions in a yeast two-hybrid assay (50).Core is also highly basic and binds RNA (19, 57), and thisassociation is dependent on the basic N terminus (57). Becauseinteractions of core with cellular proteins can be studied inmany cultured cell lines, much is known about these interac-tions, including which domains of core are required. However,due to the lack of systems for studying assembly in a quanti-tative manner, no systematic mutational analysis of core hasbeen performed to define domains that are important for itsability to assemble into an HCV capsid.

We have recently established a cell-free system (CFS) thatsupports robust HCV core assembly and forms bona fide HCVcapsids, as demonstrated by velocity sedimentation, buoyantdensity, and transmission electron microscopic analyses (28).Thus, HCV capsids produced in this system are nearly indis-tinguishable from authentic capsids isolated from the serum ofinfected patients (28). This system links de novo translation ofcore to HCV capsid assembly in eukaryotic extracts, recapitu-lating events beginning with synthesis of HCV core through thecompletion of capsid assembly. Additional advantages and fea-tures of this system are that it is amenable to quantitation,highly reproducible, can be manipulated, and supports synthe-sis of mutant core constructs. Previously we used this system todefine characteristics and requirements of HCV assembly andshowed the importance of the N-terminal RNA binding regionfor capsid assembly (28). Here we use this permissive HCVcapsid assembly system to further characterize important res-idues in the N terminus and to examine domains elsewhere inthe core protein that may be involved in capsid assembly. Wefind that clusters of basic charges are critical for HCV assemblyand that certain uncharged regions in both the N terminus andthe remainder of the protein are largely dispensable for HCVcapsid assembly.

MATERIALS AND METHODS

DNA plasmids. Cell-free plasmid vectors were derived from SP64 vector (Pro-mega) into which the 5� untranslated region (UTR) of Xenopus laevis globin hadbeen inserted at the HindIII site (44). The parental plasmid used for this studycontained the HCV core coding region from an HCV 1b isolate as describedpreviously (28). Using the parental plasmid as the template, mutant core con-structs were prepared by either site directed mutagenesis (Stratagene), or bytwo-step-PCR and cloned into the BglII and EcoRI sites of the parental plasmid(data not shown). All coding regions were verified by sequencing.

In vitro transcription and cell-free translation/assembly. In vitro transcriptionwas performed using the SP6 polymerase (New England Biolabs) and the SP64expression plasmids described above. Resulting transcripts (or transcriptionbuffer for mock transcript, as indicated) were used to program cell-free transla-tion and assembly reactions for 120 min, either using wheat germ extracts at 26°Cor using rabbit reticulocyte lysate at 37°C. Reactions were radiolabeled using[35S]methionine (ICN Biochemicals), as described previously (18, 33, 52).

Gradient analysis of cell-free reactions and cellular lysate. Calibration ofgradients to determine S value positions has been described previously (32, 33).Ten to 20 microliters of cell-free assembly reactions, diluted into 200 �l finalvolume containing 0.625% NP-40 detergent, 10 mM Tris-acetate, pH 7.4, 50 mMKAc, 100 mM NaCl, and 4 mM Mg acetate, were layered onto gradients con-taining sucrose prepared in the same buffer. Velocity sedimentation was per-formed on step gradients containing 400 �l each of 10%, 20%, 30%, and 40%

sucrose with a 200-�l 50% sucrose cushion. Centrifugation of velocity sedimen-tation gradients was performed at 201,000 � g for 55 min at 4°C in a TLS55 rotor(Beckman Coulter Optima Max centrifuge), and 200-�l fractions were collectedserially from the top. Equivalent aliquots of each gradient fraction were trichlo-roacetic acid (TCA) precipitated and subjected to sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography (AR).

Chymotrypsin digestions. Chymotrypsin digestions were done as previouslyreported (30). Either fractions 1 and 2 or fractions 6 and 7 from velocity sedi-mentation gradients were pooled and then added to chymotrypsin reactions.Chymotrypsin reactions were set up on ice and contained 300 �l of sample, 50mM Tris, pH 8.0, 20 mM CaCl2, and 1 ng/�l chymotrypsin (Bovine Pancreas;Calbiochem) in a volume of 1.2 ml. At the indicated time points, 200 �l of thereaction were removed, quenched with TCA, TCA precipitated, and analyzed bySDS-PAGE and AR. The concentration of chymotrypsin was decreased 10-foldfrom the previous report (30) to compensate for greatly reduced amount ofsubstrate in our samples.

Electron microscopy. Cell-free reactions were subjected to velocity sedimen-tation centrifugation, and fractions 6, 7, and 8 were pooled and dialyzed (mo-lecular weight cutoff, 55,000) for 1 h against phosphate-buffered saline (PBS) atroom temperature. Dialyzed samples were then settled onto carbon-coated grids(Ted Pella) for 2 to 3 min, stained with 1% uranyl acetate for 30 s, and visualizedusing a JEOL 1010 transmission electron microscope. An experienced electronmicroscopist identified and examined reactions and controls in single-blindedfashion in three separate experiments. Histograms were prepared by measuringthe diameters of all capsids in three to six fields (containing �120 capsids).Criteria for excluding small particles that were not capsids have been describedpreviously (28).

Quantitation. Autoradiographs were digitized using an AGFA Duoscan T1200scanner and Adobe Photoshop 5.5 software (Adobe Systems Incorporated).Mean band densities were determined and adjusted for band size and back-ground. Amount of assembly was quantitated as the amount of core present infractions 6, 7, and 8.

Statistics. P values were obtained using a paired student t test (one-tailed).

RESULTS

Internal deletions within the N-terminal 42 amino acidsdiminish HCV capsid assembly. We previously established aCFS that supports HCV capsid assembly and demonstratedthat approximately 60 to 80% of newly synthesized HCV corepolypeptides assemble into �100S complexes that migrate infractions 6 to 8 on our velocity sedimentation gradients. The�100S complexes correspond to HCV capsids by multiple cri-teria (28). Additionally, we showed that HCV capsids are het-erogeneous in size as is seen in vivo, and the �100S cell-freecapsids range from 75S to 120S upon analysis in higher reso-lution gradients (28). In our previous study, we demonstratedthat both forms of full-length HCV core that are found in vivo(C191, which includes the E1 signal sequence, and the mature,signal-cleaved version of core, termed C173) assemble to anequivalent extent. Furthermore, there was no difference inassembly when wheat germ extract (WG) versus rabbit reticu-locyte lysate (RRL) was utilized as a source of cellular factorsfor the CFS (28). Thus, unless otherwise noted, experiments inthe current study were performed in the WG CFS using C173as wild-type (WT) core, with % assembly defined as % ofradiolabeled core polypeptides present in gradient fractions 6to 8.

Additionally, in the previous study, we showed that thehighly basic N-terminal 68 amino acids of HCV core containresidues important for HCV capsid assembly, since N-terminaltruncations of HCV core altered assembly while truncations inthe C-terminal half of core had no effect. We found that de-leting the N-terminal 10 amino acids (�N10) had no effect oncell-free assembly (79% assembly) but deleting the N-terminal42 (�N42) or 68 (�N68) amino acids decreased assembly to

VOL. 79, 2005 HCV CORE RESIDUES CRITICAL FOR HCV CAPSID ASSEMBLY 6815

12% and 8%, respectively (28). Qualitatively this was seen invelocity sedimentation profiles in which WT C173 migratedlargely in fractions 6 to 8, and �N42 and �N68 migrated at thetop of the gradient in fractions 1 and 2, representing complexesof 10S (28).

Based on these observations, we wanted to further identifyspecific residues or motifs that are important for capsid assem-bly. Therefore, we generated a series of internal deletionswithin the N-terminal 42 amino acids (Fig. 1A). When totaltranslations were analyzed by SDS-PAGE, we found that allthe deletion mutants expressed to similar levels as WT C173and migrated at their expected sizes (Fig. 1B). We then testedtheir ability to assemble into HCV capsids in the CFS byvelocity sedimentation (Fig. 1C), as described previously (28).Because core is known to be extremely lipophilic, the velocitysedimentation gradients contained 0.625% NP-40 to solubilizenonspecific proteins aggregating with core as well as associat-

ing membranes, allowing the true sedimentation value of HCVcore and assembled HCV capsids to be assessed. Deletion of32 amino acids (designated �11-42) resulted in a statisticallysignificant decrease to 21% assembly, compared to WT C173,which yielded 60% assembly. This was consistent with resultsobtained with the previously described N-terminal truncationmutants (28) and confirmed that residues required for assem-bly are present between amino acids 11 to 42. However, wefound that smaller deletions that removed 11 to 22 residueswithin this region (�11-21, 21-42, and 31-42) inhibited assem-bly to only a modest extent (Fig. 1C), suggesting that thisregion does not contain a single discrete motif that is criticalfor assembly.

Basic residues in the N terminus of core are critical for HCVcapsid assembly. Given these findings, we hypothesized thatresidues important for assembly are likely to be dispersedbetween amino acids 11 to 42 and might function coopera-tively. The N terminus contains two regions with a high densityof basic amino acids linked by a neutral region of 15 aminoacids (Fig. 2A). Therefore, we hypothesized that the two basicregions and possibly the neutral linking region are importantfor HCV assembly. To test this possibility, we constructedtargeted deletions of the two basic clusters (�1 and �2) and theneutral linker (�L). The �1 construct (amino acids 8 to 25deleted) removed 7 basic residues, while �2 (amino acids 39 to64 deleted) removed 10 basic residues, and �L (amino acids 27to 38 deleted) removed no basic residues (Fig. 2A). The dele-tion mutants expressed to similar levels and migrated on SDS-PAGE at their expected sizes (Fig. 2B). When we analyzedtheir ability to assemble, we found that deleting either clusterof basic amino acids (�1 or �2) diminished the ability of coreto assemble to approximately 25%. Surprisingly, deleting theentire neutral region (�L) between the basic clusters had noeffect on HCV capsid assembly in the CFS (Fig. 2C and D).

Next, we directly assessed the contribution of the basic res-idues in the N terminus by substituting alanines for basic res-idues in the N terminus. Fifteen mutants encoding as few asone and as many as 16 basic residue substitutions in one orboth basic clusters were constructed (Fig. 3A). We found thatthe ability of the mutant constructs to assemble in the CFSdecreased as more basic residues were mutated (Fig. 3B andC). When only one basic residue was mutated (RK1A andRK1B, Fig. 3) there was little or no effect on assembly (�60%assembly for each construct compared to 61% assembly forWT core). However, as the number of basic residues mutatedincreased, a progressive decrease in the ability of core to as-semble was observed (i.e., �48% for RK2A and RK2B; �30%for RK3A and RK3B; �25% for RK4A and RK4B), suggest-ing that basic charge in this region is critical for capsid assem-bly. It should be noted that not all mutants expressed as well asWT (Fig. 3B, line graph), but this likely does not account forthe lack of assembly because we have previously demonstratedthat HCV assembly is minimally dependent on core concen-tration in the CFS (28). Consistent with this observation, whenWT core expression was lowered to less than that of RK4B,were observed (data not shown). Additionally, some basiccharge substitution mutants displayed equivalent amounts oftranslation relative to WT C173 but had significantly reducedlevels of assembly (i.e., RK6, RK9, RK13, and RK16 in Fig.

FIG. 1. N-terminal deletions in HCV core reduce HCV capsid as-sembly. (A) Schematic representation of the deletions made comparedto WT C173. Numbers indicate amino acid number in WT HCV core.(B) Cell-free reactions using WG extracts were programmed withtranscripts encoding WT C173 or the indicated mutants. Aliquots wereanalyzed by SDS-PAGE and autoradiography. A representative auto-radiograph (AR) shows similar amounts of translation in equal ali-quots of reactions programmed with the different constructs and theexpected differences in migration by SDS-PAGE. (C) Cell-free reac-tions were programmed with the indicated constructs and analyzed byvelocity sedimentation. The % assembly of core (i.e., core migrating infractions 6, 7, and 8 as % of total core synthesized for each reaction)is shown as a bar graph. Total amount of translation for each constructwas also measured by densitometry (arbitrary units) and is shown by aline graph (right axis). The number of amino acids deleted in eachconstruct is indicated in parentheses. Error bars represent standarderror of the mean from three independent experiments. Constructsthat demonstrate a statistically significant reduction in assembly rela-tive to WT C173 are indicated (*, P 0.01).

6816 KLEIN ET AL. J. VIROL.

3B). These findings are consistent with basic charge in thisregion being required for efficient capsid assembly.

Table 1 summarizes all the constructs encoding N-terminalmutations (truncations, deletions, and substitutions) that wehave studied in the CFS and is organized by the number ofbasic residues that are altered. The amount of assembly foreach construct is indicated as % of WT core assembly (withWT assembly set at 100%). This comparison reveals that re-moval or mutation of one or two basic residues was generallywell tolerated, while further increasing the number of basicresidues progressively diminished HCV capsid assembly. Theapparent correlation between the number of basic residuespresent in the N terminus and the ability of HCV core toassemble suggests that these basic residues are an importantdeterminant of HCV capsid assembly.

HCV core polypeptides encoding mutations form capsidsthat are stable. We repeatedly observed that core mutantsdisplaying intermediate levels of assembly migrated differentlyin velocity sedimentation gradients compared to fully assem-bly-competent or assembly-incompetent constructs. In addi-tion to being present in the assembled fractions, core polypep-tides that assembled to intermediate levels were dispersedthroughout the gradient (i.e., �1 and �2 in Fig. 2D) rather than

solely forming peaks in specific fractions as was the case forassembly-competent (i.e., fractions 6 and 7 for WT C173 in Fig.2D) or assembly-incompetent mutants (i.e., fractions 1 and 2for �N68 in Fig. 6B). This led us to ask whether constructs thatassemble to intermediate levels form unstable complexes thatgive rise to the more dispersed migration pattern during thevelocity sedimentation analysis. We examined stability by iso-lating capsids (in fractions 6 and 7) formed by C173 or coremutants as well as other complexes (in fractions 3 and 4)formed by core mutants that display a limited level of assem-bly. We then subjected these complexes to a second round ofvelocity sedimentation on the same type of gradients. If thecapsids and/or complexes are inherently stable, they shouldmigrate in the same fractions upon repeat velocity sedimenta-tion. Capsids of WT C173 and two mutants (�2 and RK7) werestable, since they migrated to fractions 6 and 7 on the secondvelocity sedimentation gradient (Fig. 4). RK7 did appear tospread out somewhat during the second velocity sedimentationanalysis but mostly migrated in fractions 6 and 7. In contrast,when smaller complexes (i.e., fractions 3 and 4) of the twomutants �2 and RK7 were reanalyzed by velocity sedimenta-tion, they migrated mostly to the top of the gradient (fraction1, which contains monomers and complexes of 10S), suggest-

FIG. 2. Deletion of basic clusters inhibits HCV capsid assembly. (A) Schematic representation of the deletion constructs compared to WTC173. Dashes indicate any amino acid other than arginine (R) or lysine (K). Numbers indicate amino acid number in WT HCV core. (B) Cell-freereactions using WG extracts were programmed with transcripts encoding WT C173 or the indicated mutants. Aliquots were analyzed bySDS-PAGE and autoradiography. A representative AR shows similar amounts of translation for the different constructs and the expecteddifferences in migration. (C) The % assembly for each construct was calculated as in Fig. 1C and is shown as a bar graph (left axis). Total amountof translation for each construct was also measured by densitometry (arbitrary units) and is shown by a line graph (right axis). Error bars representstandard error of the mean from three independent experiments. Constructs that demonstrate a statistically significant reduction in assemblyrelative to WT C173 are indicated (**, P 0.001). (D) Representative AR from the velocity sedimentation analysis. Numbers above the lanesrepresent fraction number (fraction 1 corresponds to �10S complexes at the top of the gradient), and bar indicates fractions containing HCVcapsids (�100S).


ing that they lack stability. Thus, it appears that some mutantconstructs that assemble to intermediate or low levels form atleast two types of complexes: stable capsid-like complexes thatmigrate in the �100S assembled fractions and complexes thatmigrate in other regions of the gradient that are unstable.

Unassembled core polypeptides are sensitive to chymotryp-sin, unlike assembled capsids. To further address whethermutant HCV core constructs assemble into capsids at a lowlevel in the cell-free system, we modified a previously described

assay that was used to demonstrate that assembled recombi-nant HCV core is resistant to chymotrypsin digestion, whereasunassembled recombinant HCV core is relatively susceptibleto chymotrypsin digestion (30). If the mutant complexes infractions 6 and 7 are intact capsids, they should be resistant tochymotrypsin digestion. To test this, we isolated �100S capsidlike complexes, as well as unassembled HCV core, from cell-free reactions expressing WT or mutant constructs and sub-jected them to chymotrypsin digestion. Figure 5 shows that WT

FIG. 3. Mutation of four or more basic amino acids in HCV core reduces HCV capsid assembly. (A) Schematic representation of the pointmutations made in the N-terminal 68 amino acids compared to WT C173. Dashes indicate any amino acid other than arginine or lysine; mutatedamino acids are in bold. Numbers indicate amino acid number in WT HCV core. (B) The % assembly for each construct was calculated as in Fig.1C and is shown as a bar graph. Total amount of translation for each construct was also measured by densitometry and is shown by a line graphas in Fig. 1C. Error bars represent standard error of the mean from three independent experiments. Constructs that demonstrate a statisticallysignificant reduction in assembly relative to WT C173 are indicated (*, P 0.01; **, P 0.001). (C) Representative AR of the velocitysedimentation analysis of selected constructs. Numbers above the lanes represent fraction number (fraction 1 corresponds to the top of thegradient), and bar indicates fractions containing HCV capsids.


core from the assembled fractions 6 and 7 (right panels) wasrelatively resistant to chymotrypsin, while the very smallamount of WT core present in the top two fractions (leftpanels) was protease sensitive. Both the protease sensitivity ofunassembled HCV core and the protease resistance of assem-bled capsids in the CFS resembled the findings previouslyreported for unassembled and assembled recombinant HCVcore (30). Similarly, despite different input amounts for differ-ent constructs, mutant core present in unassembled top frac-tions was relatively protease sensitive while core from assem-bled fractions 6 and 7 was relatively protease resistant for allconstructs examined, including an assembly-competent mutantconstruct (RK2B), mutant constructs with basic amino acidsmutated in either the first or second cluster (RK4B and RK6,respectively), and a construct containing a large deletion (�2)(Fig. 5). Thus, the protease resistance of the small amounts of�100S complexes formed by mutant constructs supports theconclusion that these constructs undergo a limited amount ofcapsid assembly. Additionally, the examined protease sensitiv-ity of the unassembled fractions suggests that the mutationsare unlikely to grossly affect protein folding, although it isimpossible to rule out very subtle effects on folding.

Wild-type core does not rescue assembly-defective mutants.In certain systems it is possible to rescue defective mutants byexpressing a competent construct in trans. For example, bud-ding-defective human immunodeficiency virus type 1 (HIV-1)constructs that are missing the p6 domain of the HIV-1 capsid

protein can be complemented in trans in cells with constructsthat encode regions of the p6 domain (40). In the case ofHIV-1 capsid assembly, assembly-defective nonmyristoylatedcapsid precursor GagPol proteins can be rescued by coexpres-sion of myristoylated assembly-competent capsid proteins (60).Conversely, mutant capsid proteins can interfere with the func-tion of WT proteins by acting as dominant-negative mutants, asshown by studies of specific mutations in HIV-1 Gag (14).Together, these data indicate that in the case of HIV-1 capsidformation, capsid proteins produced from different transcriptsinteract during the assembly process. To test whether WTC173 rescues the assembly of assembly-defective constructs orwhether core mutants act in a dominant-negative manner toinhibit assembly of WT C173, we programmed cell-free reac-tions with equivalent amounts of transcripts encoding WT and

FIG. 4. Capsids composed of mutant core proteins are stable, whileother complexes composed of core mutants are not. Cell-free reactionscontaining wheat germ (WG) were programmed with transcripts en-coding WT C173 (A), �2 (B), or RK7 (C) and analyzed by velocitysedimentation. Fractions 6 and 7 (A, B, and C) or fractions 3 and 4 (Band C) were pooled and were subjected to velocity sedimentation fora second time on the same type of gradient as indicated by bar witharrow pointing to repeat sedimentation. Top AR in A, B, and C showthe first velocity sedimentation gradients; middle AR in B and C showresults of repeat velocity sedimentation of fractions 3 and 4 as indi-cated by arrow; and bottom AR in A, B, and C show results of repeatvelocity sedimentation of fractions 6 and 7 as indicated by arrow.Numbers above the lanes represent fraction number (fraction 1 cor-responds to the top of the gradient). Experiment was repeated threetimes with one representative experiment shown.

TABLE 1. Summary of constructs encoding mutations in the Nterminus of HCV core

Construct No. of RKmutationsa Assemblyb Del/Subc Figured

WT C173 0 100�L 0 101 Del 2RK1A 1 105 Sub 3RK1B 1 92 Sub 3�31-42 2 57 Del 1RK2A 2 88 Sub 3RK2B 2 72 Sub 3�N10 3 106 Del *�21-42 3 79 Del 1RK3A 3 50 Sub 3RK3B 3 55 Sub 3�11-21 4 63 Del 1RK4A 4 41 Sub 3RK4B 4 29 Sub 3RK6 6 27 Sub 3�N20 7 40 Del *�11-42 7 33 Del 1RK7 7 29 Sub 3�1 8 33 Del 2�N30 8 30 Del *RK8 8 29 Sub 3RK9 9 20 Sub 3�2 10 36 Del 2�N42 10 15 Del *RK13 13 20 Sub 3RK16 16 19 Sub 3�N68 19 10 Sub *

a Number of basic residues mutated or deletedb % Assembly of WT C173 (WT C173 100%).c Del, deletion; Sub, alanine substitution.d Figure in this study showing analysis of the mutant construct. *, Figure shown

in Klein et al. (28).


mutant constructs (�N68, �11-42, or �N2). These constructsmigrate at different positions on SDS-PAGE from WT C173(Fig. 6A) and therefore can be distinguished when expressed inthe same reaction. When we analyzed their ability to assembleby velocity sedimentation, we found that WT C173 assembledto an equivalent extent in the absence or presence of mutantconstructs, suggesting that none of the mutants examined be-haved in a dominant-negative manner (compare panels in Fig.6A to C173 panel in B; graphed in C). Moreover, assembly ofthe mutant constructs did not increase when coexpressed withWT, demonstrating that WT C173 does not complement theassembly-defective mutants (compare panels in Fig. 6A tothree lower panels in B; graphed in D). Additionally, coex-pressing WT and mutant constructs did not alter the distribu-tion on velocity sedimentation gradients of the mutant coreconstructs examined, showing that formation or accumulationof other complexes was unaffected (compare Fig. 6A to B).Equivalent results were found when an assembly-competentmutant (�82-102; see Fig. 7) was coexpressed with the mutantsin Fig. 6 (data not shown).

Mutations in other regions of core do not alter HCV capsidassembly. Other groups have suggested that regions outside ofthe N terminus of core are important for HCV capsid assem-bly. Amino acids 82 to 102 were found to contain a homotypicinteraction domain by a yeast two-hybrid assay that measurescore-core interactions (50). A morphological study of capsidassembly done in BHK21 cells using a Semliki forest virusexpression system implicated an aspartic acid at amino 111 asimportant for HCV assembly (8), although it was unclear to

what extent WT core or the mutant assembled in this system.Additionally, other regions in the central region of core calleddomain 2 (42) are known to be important for targeting of theHCV core protein (24, 25, 43) but have not been examinedwith respect to capsid assembly. For example, two prolineresidues (at amino acids 138 and 143) form a proline knotmotif that is important for targeting core to lipid droplets (25).Because core–core interactions and targeting are likely to beimportant for HCV capsid formation, we wanted to quantita-tively examine what effect these mutations, shown in Fig. 7A,had on the ability of core to assemble in the cell-free system. Incontrast to the assembly-defective N-terminal mutants shownpreviously, core constructs encoding �82-102, D111A, and mu-tations in prolines 138 and 143 assembled as well as WT C173by velocity sedimentation (Fig. 7B and C). Similar results wereobtained when D111A was examined using a cell-free systemcontaining RRL rather than WG (Fig. 7B, compare right andleft sides). To confirm these findings we also subjected frac-tions containing �100S capsids encoding the D111A and �82-102 mutations to transmission electron microsocopy (TEM;Fig. 8A to C). We had previously shown that capsids formed byWT C173 in the CFS closely resemble capsids obtained frompatient serum (28). We found that the assembly-competent�82-102 and D111A mutants formed abundant numbers ofcapsids that resembled capsids produced by WT C173 in theCFS. Measurements revealed a bimodal distribution of diam-eters for capsids formed by the �82-102 mutant. This distribu-tion closely resembled the distribution of diameters displayedby WT C173 capsids prepared in parallel (Fig. 8D). In contrast,

FIG. 5. Assembled capsids display resistance to chymotrypsin proteolysis. Cell-free reactions containing wheat germ (WG) were programmedwith transcripts encoding WT C173, RK2B, RK4B, RK6, or �2 and analyzed by velocity sedimentation. Fractions 1 and 2 (unassembled core, leftpanels) or fractions 6 and 7 (assembled core, right panels) were pooled and subjected to proteolytic digestion with chymotrypsin for the indicatedtimes (minutes) following a previously reported protocol (30). Reactions were analyzed by SDS-PAGE and AR. Presented are AR showing thatunassembled core is protease sensitive and that assembled core is resistant to the same concentration of chymotrypsin for all constructs. Arrowsindicate migration of each full-length construct. Experiment was repeated three times with one representative experiment shown.


capsids encoding the D111A mutation had larger diameterswhile maintaining a bimodal distribution of diameters (Fig.8D). These data suggest that, at least in the CFS, these resi-dues are not essential for HCV capsid assembly, although theaspartic acid at position 111 does appear to influence capsidsize.

Phosphorylated serines are not essential for capsid assem-bly. The fact that cultured cells do not support HCV assemblysuggests that this process can be regulated; however, this reg-ulation has not been studied in detail. One common mecha-nism frequently used to regulate cellular processes and cellularproteins is phosphorylation. HCV core has been shown to bephosphorylated in vitro at serines at amino acids 53, 99, and116 by protein kinase A and C (58) and are phosphorylated inhuman liver cell lines (38). These serines are highly conservedacross different HCV genotypes (11), suggesting they may playan important role for HCV. Studies show that phosphorylationof core may regulate targeting of core to the nucleus (38), andthe ability of HCV core to suppress HBV transcription (58).Whether these phosphorylated serine residues play a role incapsid assembly has not been addressed previously. To exam-ine this, serines at 53, 99, and 116 were sequentially convertedto alanines (Fig. 9A). Constructs encoding these mutationstranslated to an equivalent extent and assembled as well as WTC173 in both WG and RRL cell extracts (Fig. 9B and C). Thus,it appears that serines in core that are thought to undergophosphorylation are not required for HCV capsid assembly inthe cell-free system, even when a mammalian extract is used.Conversion of serine 53 to aspartic acid (S53D) to mimic thephosphorylated state also had no effect (Fig. 9B and C). Theeffect of conversion of other serines to aspartic acids has notyet been examined.

DISCUSSION

Here we have systematically analyzed regions of the HCVcore protein that are important for capsid assembly using arecently described HCV cell-free system that allows for a quan-titative analysis of assembly in a cellular context. Because itsupports efficient capsid assembly (28), this system differs fromalmost all reported cellular systems in which HCV core fails toassemble into capsids. Because of the lack of systems for study-ing HCV capsid assembly, surrogate assays for assembly, suchas core–core interactions in yeast two-hybrid assays and non-quantitative assays, have been used to define residues thatappeared to be important for HCV assembly. It has beendifficult to fully interpret results from these studies, leaving agap in our understanding of which residues and domains are

FIG. 6. WT C173 does not rescue nonassembling mutant con-structs and mutants do not behave as dominant negatives. Two sets ofcell-free cotranslation reactions, performed in parallel and analyzed byvelocity sedimentation and SDS-PAGE, are shown as ARs (A, B)which were used to quantitate % assembly (C and D). (A) Shown arevelocity sedimentation analyses of the first set of cotranslations, pro-grammed with WT C173 transcript and either �N68, �11-42, or �2transcript. (B) Shown are velocity sedimentation analyses of the par-allel control cotranslation reactions, programmed with WT C173,�N68, �11-42, or �2 transcript, along with mock transcript. Positionsof WT and mutant constructs in SDS-PAGE gels are indicated to theright. Numbers above the lanes represent fraction number (fraction 1

corresponds to the top of the gradient), and bar indicates fractionscontaining HCV capsids. Note that aliquots of total translation(T) were included at both ends of the AR as a reference for positionof the different constructs on SDS-PAGE. (C) The % assembly of WTC173 in each cotranslation from A and B that contains C173 wasquantitated as in Fig. 1C and is shown as a bar graph. (D) The %assembly of each core mutant from A and B when cotranslated withmock transcript (dark shading) or with WT C173 (light shading) wasquantitated as in Fig. 1C and is shown as bar graphs. Error barsrepresent standard error of the mean from three independent exper-iments.


important for HCV assembly. Here we showed that the N-terminal basic region plays a key role in HCV assembly in theCFS. Interestingly, this region is also known to bind RNA,which is thought to nucleate capsid assembly for a number ofviruses (5, 12, 13, 55, 62). Conversely, we found that regionsoutside the N terminus (i.e., distal to amino acid 82) did notinfluence HCV capsid assembly per se.

The N terminus of HCV core contains two highly conservedclusters of basic residues separated by a neutral linker region(11). Within the N terminus, the most important residues forassembly appeared to be the basic residues, because deletingthe entire neutral linker did not affect HCV assembly. Trun-cations, deletions, and point mutants encompassing basic res-idues diminished the ability of core to assemble. Analyzing allof these mutants together revealed that removing more thanfour basic residues severely impaired the ability to assemble ina cell-free system containing WG extract (Table 1). Our dataalso suggest that no specific motif for assembly exists becausemutating several basic amino acids, regardless of their exactlocation within the two N-terminal basic clusters, effectivelyinhibited HCV assembly (see Table 1). These data suggest thatthe overall charge of the N terminus is important for HCVassembly; however, we cannot rule out that specific residues inthis region are more important than others for HCV capsidassembly. Additionally, the contribution of many of the un-charged amino acids has not been addressed. Note that eventhough its ability to assemble is greatly diminished, the RK16mutant (encoding 16 basic to neutral amino acid substitutions)maintained some capacity to assemble (see Fig. 3B). This smallamount of residual assembly may reflect contributions of basicresidues that remain in the N terminus, contributions of basicresidues distal to the N-terminal 62 amino acids that we ex-amined, or the contribution of nonbasic residues for assembly.

Our finding that basic charge in the N terminus of HCV coreis important for HCV capsid assembly is consistent with studiesof assembly of other RNA viruses. While the effect of muta-tions in capsid proteins on assembly have not been studiedsystematically for flaviviruses, mutational analyses have beenperformed for other RNA viruses. Most extensive have beenmutational analyses of the retroviral capsid protein, Gag (6).Typically, these studies have demonstrated that basic charge inthe Gag nucleocapsid domain (NC) is important for nonspe-cific association with RNA and for assembly. For example, inthe case of HIV-1 Gag, charged residues in NC are requiredfor capsid assembly (16, 17, 56). Consistent with these findings,studies of assembly of purified retroviral capsid proteins invitro have demonstrated the importance of RNA in nucleatingassembly of capsid proteins (12, 13). Studies of alphaviruscapsid proteins have also demonstrated the importance of ba-sic residues for RNA encapsidation (61). However, the role ofbasic charge in promoting capsid formation remains less clearfor alphaviruses than for retroviruses. Studies of purified al-phavirus capsid proteins in vitro showed that nonspecific asso-ciation with RNA is important for nucleating assembly of al-phavirus core proteins (49, 62, 63). A study of Semliki Forestvirus capsid protein mutants expressed in mammalian cellsfound that complete deletion of the basic charged region in theN terminus eliminated assembly, but large partial deletionswere tolerated (20). In contrast, deletions in the Ross Rivervirus N terminus that eliminated most of the basic residues did

not impair capsid protein interactions or virus particle produc-tion, although viral RNA content was drastically reduced (22).Nevertheless, most studies of RNA virus assembly support amodel in which regions of basic charge in capsid proteinsinteract nonspecifically with RNA, which in turn promotesassembly. Data presented here for HCV capsid assembly fitthis model. The exact mechanism by which basic charge acts to

FIG. 7. Mutations outside of the N terminus do not affect HCVassembly. (A) Schematic representation of the mutations analyzed inthis figure compared to WT C173. Numbers indicate amino acid num-ber in WT HCV core. (B) Cell-free reactions containing either WG orRRL were programmed with the indicated constructs. Reactions wereanalyzed by velocity sedimentation, SDS-PAGE, and autoradiography.The amount of assembly of each mutant relative to WT C173 wasquantitated and is shown as a bar graph. (C) Cell-free reactions con-taining WG were programmed with constructs encoding single (P138Aand P143A) or double (P138/143A) mutations at specific proline res-idues. Reactions programmed with WT C173 and �N68 were includedas positive and negative controls, respectively. Reactions were ana-lyzed by velocity sedimentation, SDS-PAGE, and autoradiography.The % assembly was quantitated as in Fig. 1C and is shown as a bargraph. Total amount of translation for each construct was measuredand is shown by a line graph, as in Fig. 1C. Error bars representstandard error of the mean from three independent experiments.


promote assembly remains unclear, but the identification ofkey residues presented here will allow future studies to addressthis question.

We also investigated whether expressing WT or mutant coreconstructs together would alter assembly of either construct.Surprisingly, WT C173 failed to rescue any mutants tested, nordid any of the mutant constructs examined act in a dominant-negative manner, suggesting that the core encoded by separateconstructs are unable to interact. This is consistent with thepossibility that there exist microenvironments where assemblyoccurs and that these microenvironments contain only onetype of core polypeptide. If such microenvironments contain asingle mRNA, core polypeptides translated from that particu-lar mRNA might be preferentially incorporated together intocapsids and fail to interact with core polypeptides translatedfrom a different mRNA. Such close coordination of translationwith assembly could explain the failure of WT C173 to com-plement core mutants in our experiments and would also beconsistent with our previous findings that HCV assembly oc-curs very rapidly in pulse-chase experiments and is relativelyindependent of core polypeptide concentration (28). However,this model remains to be tested directly.

Morphological studies of core assembly and two-hybrid as-says for core-core interactions have been used in the past toevaluate mutations in HCV core. When a construct encoding adeletion in a domain thought to be involved in homotypicinteractions (amino acids 82 to 102) (50) was programmed intoour cell-free assembly system, we observed no difference in itsability to assemble relative to WT C173. This region, which isthought to facilitate core dimerization (50), may not be essen-tial for assembly but instead may be important for core tointeract with cellular factors that regulate other functions. Wealso found that converting an aspartic acid to alanine at aminoacid 111 (D111A) had no effect on HCV capsid assembly, incontrast to another study that implicated a role for the asparticacid at 111 in HCV assembly (8). The amount of assembly inthe earlier study was not quantified, so the severity of thereported defect is unclear. Interestingly, the D111A mutationin core creates a PSAP motif, which is known to facilitatebudding for other viruses through interaction with TSG101 andESCRT proteins (21). Thus, it is possible that the D111Amutation causes core to interact with cellular machinery suchas TSG101 or ESCRT proteins, which may mistarget core andthereby inhibit the ability of core to assemble properly in intactcells, but this hypothesis remains to be tested.

It is likely that interaction of core with other cellularfactors plays a role in regulating its ability to assemble. Forexample, core is known to target to lipid droplets in mam-malian cells (1, 24, 25, 43, 53), and this is dependent onproline residues located in the C terminus of core. Whenprolines at amino acids 138 and 143 are mutated, core nolonger targets to lipid droplets, and these core mutants are

FIG. 8. Transmission electron microscopy shows similar morphol-ogy for capsids formed by WT and assembly-competent mutants. (A,B, and C) Cell-free reactions programmed with the indicated con-structs (A, C173; B, D111A; C, �82-102) were separated by velocity

sedimentation, and fractions 6, 7, and 8 (containing �100S capsids)were pooled, dialyzed against PBS, and subjected to negative staintransmission electron microscopy (TEM). Scale bars represent 100 nm.Shown are representative results from 3 independent experiments.(D) Histograms of particle sizes for each of the indicated constructs,with �120 capsids counted for each construct.


degraded (25). When core constructs containing these samemutations were expressed in the CFS, they were stable andassembled into capsids to the same extent as WT core. Thisunderscores the need to take into account that althoughcell-free systems faithfully recapitulate many cellular pro-cesses, the CFS described here may not reproduce some ofthe regulatory events that occur in human liver cells, thenatural target of HCV. This dissociation of HCV capsidassembly in the CFS from negative (or positive) regulatoryprocesses is useful because it allows assembly to be studiedindependently. However, this caveat must be kept in mindwhen interpreting the assembly data. Note that more com-plex cell-free systems can be devised to reconstitute regula-tory events associated with assembly. Indeed, we have foundthat by adding liver cell lysates to cell-free reactions, WTHCV capsid assembly is partially inhibited, suggesting thatin the presence of specific mammalian cellular factors, HCVassembly can be down-regulated (28). While the criticalresidues for HCV assembly appear to reside at the N ter-minus, it is possible that other domains in the core protein(i.e., the C terminus and the E1 signal sequence) play im-portant roles in regulating HCV assembly in vivo. It will beinteresting to determine whether mutations in these regions

alter the ability of mammalian cell lysates to inhibit HCVcapsid assembly in the CFS.

ACKNOWLEDGMENTS

This work was supported by a pilot project grant to J.R.L. fromPuget Sound Partners (#26145) and an NIH training grant award toK.C.K. (NIH T32 CA09229).

RRL was a gift from V. Lingappa at the University of Californiaat San Francisco. We thank L. Caldwell at the Fred HutchinsonCancer Research Center for assistance with electron microscopy; L.Walker for technical assistance; and J. Dooher, V. Lingappa, M.Linial, M. Newman, M. Orr, J. Overbaugh, and L. Walker forhelpful discussions.

REFERENCES

1. Barba, G., F. Harper, T. Harada, M. Kohara, S. Goulinet, Y. Matsuura, G.Eder, Z. Schaff, M. J. Chapman, T. Miyamura, and C. Brechot. 1997. Hep-atitis C virus core protein shows a cytoplasmic localization and associates tocellular lipid storage droplets. Proc. Natl. Acad. Sci. USA 94:1200–1205.

2. Bartenschlager, R., and V. Lohmann. 2001. Novel cell culture systems for thehepatitis C virus. Antiviral Res. 52:1–17.

3. Bartenschlager, R., and V. Lohmann. 2000. Replication of hepatitis C virus.J. Gen. Virol. 81(Pt. 7):1631–1648.

4. Baumert, T. F., S. Ito, D. T. Wong, and T. J. Liang. 1998. Hepatitis C virusstructural proteins assemble into viruslike particles in insect cells. J. Virol.72:3827–3836.

5. Beckett, D., H. N. Wu, and O. C. Uhlenbeck. 1988. Roles of operator andnon-operator RNA sequences in bacteriophage R17 capsid assembly. J. Mol.Biol. 204:939–947.

FIG. 9. Serines that are phosphorylated in cells are not essential for capsid assembly in the CFS. (A) Schematic representation of the mutationsanalyzed in this figure compared to WT C173. Numbers indicate amino acid number in WT HCV core. (B) Cell-free reactions containing WG orRRL (as indicated) were programmed with the indicated constructs. The amount of assembly of each mutant as % of WT C173 assembly is shownas a bar graph. Total amount of translation for each construct was also measured by densitometry and is shown by a line graph as in Fig. 1C. Errorbars represent standard error of the mean from three independent experiments. (C) Representative AR of the velocity sedimentation analysis ofselected reactions. Numbers above the lanes represent fraction number (fraction 1 corresponds to the top of the gradient), and bar indicatesfractions containing HCV capsids.


6. Berkowitz, R., J. Fisher, and S. P. Goff. 1996. RNA packaging. Curr. Top.Microbiol. Immunol. 214:177–218.

7. Blanchard, E., D. Brand, S. Trassard, A. Goudeau, and P. Roingeard. 2002.Hepatitis C virus-like particle morphogenesis. J. Virol. 76:4073–4079.

8. Blanchard, E., C. Hourioux, D. Brand, M. Ait-Goughoulte, A. Moreau, S.Trassard, P. Y. Sizaret, F. Dubois, and P. Roingeard. 2003. Hepatitis Cvirus-like particle budding: role of the core protein and importance of itsAsp111. J. Virol. 77:10131–10138.

9. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation ofHCV RNA replication in cell culture. Science 290:1972–1974.

10. Blight, K. J., J. A. McKeating, J. Marcotrigiano, and C. M. Rice. 2003.Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture.J. Virol. 77:3181–3190.

11. Bukh, J., R. H. Purcell, and R. H. Miller. 1994. Sequence analysis of the coregene of 14 hepatitis C virus genotypes. Proc. Natl. Acad. Sci. USA 91:8239–8243.

12. Campbell, S., and A. Rein. 1999. In vitro assembly properties of humanimmunodeficiency virus type 1 Gag protein lacking the p6 domain. J. Virol.73:2270–2279.

13. Campbell, S., and V. M. Vogt. 1995. Self-assembly in vitro of purified CA-NCproteins from Rous sarcoma virus and human immunodeficiency virus type1. J. Virol. 69:6487–6497.

14. Chazal, N., B. Gay, C. Carriere, J. Tournier, and P. Boulanger. 1995. Humanimmunodeficiency virus type 1 MA deletion mutants expressed in baculov-irus-infected cells: cis and trans effects on the Gag precursor assembly path-way. J. Virol. 69:365–375.

15. Chen, C. M., L. R. You, L. H. Hwang, and Y. H. Lee. 1997. Direct interactionof hepatitis C virus core protein with the cellular lymphotoxin-beta receptormodulates the signal pathway of the lymphotoxin-beta receptor. J. Virol.71:9417–9426.

16. Cimarelli, A., S. Sandin, S. Hoglund, and J. Luban. 2000. Basic residues inhuman immunodeficiency virus type 1 nucleocapsid promote virion assemblyvia interaction with RNA. J. Virol. 74:3046–3057.

17. Dawson, L., and X. F. Yu. 1998. The role of nucleocapsid of HIV-1 in virusassembly. Virology 251:141–157.

18. Erickson, A. H., and G. Blobel. 1983. Cell-free translation of messengerRNA in a wheat germ system. Methods Enzymol. 96:38–50.

19. Fan, Z., Q. R. Yang, J. S. Twu, and A. H. Sherker. 1999. Specific in vitroassociation between the hepatitis C viral genome and core protein. J. Med.Virol. 59:131–134.

20. Forsell, K., M. Suomalainen, and H. Garoff. 1995. Structure-function rela-tion of the NH2-terminal domain of the Semliki Forest virus capsid protein.J. Virol. 69:1556–1563.

21. Freed, E. O. 2002. Viral late domains. J. Virol. 76:4679–4687.22. Frolov, I., E. Frolova, and S. Schlesinger. 1997. Sindbis virus replicons and

Sindbis virus: assembly of chimeras and of particles deficient in virus RNA.J. Virol. 71:2819–2829.

23. Gale, M., Jr., and M. R. Beard. 2001. Molecular clones of hepatitis C virus:applications to animal models. Ilar J. 42:139–151.

24. Hope, R. G., and J. McLauchlan. 2000. Sequence motifs required for lipiddroplet association and protein stability are unique to the hepatitis C viruscore protein. J. Gen. Virol. 81:1913–1925.

25. Hope, R. G., D. J. Murphy, and J. McLauchlan. 2002. The domains requiredto direct core proteins of hepatitis C virus and GB virus-B to lipid dropletsshare common features with plant oleosin proteins. J. Biol. Chem. 277:4261–4270.

26. Hsieh, T. Y., M. Matsumoto, H. C. Chou, R. Schneider, S. B. Hwang, A. S. Lee,and M. M. Lai. 1998. Hepatitis C virus core protein interacts with heteroge-neous nuclear ribonucleoprotein K. J. Biol. Chem. 273:17651–17659.

27. Ikeda, M., M. Yi, K. Li, and S. M. Lemon. 2002. Selectable subgenomic andgenome-length dicistronic RNAs derived from an infectious molecular cloneof the HCV-N strain of hepatitis C virus replicate efficiently in culturedHuh7 cells. J. Virol. 76:2997–3006.

28. Klein, K. C., S. J. Polyak, and J. R. Lingappa. 2004. Unique features ofhepatitis C virus capsid formation revealed by de novo cell-free assembly.J. Virol. 78:9257–9269.

29. Kountouras, J., C. Zavos, and D. Chatzopoulos. 2003. Apoptosis in hepatitisC. J. Viral Hepat. 10:335–342.

30. Kunkel, M., and S. J. Watowich. 2002. Conformational changes accompa-nying self-assembly of the hepatitis C virus core protein. Virology 294:239–245.

31. Lindsay, K. L. 2002. Introduction to therapy of hepatitis C. Hepatology36:S114—S120.

32. Lingappa, J. R., R. L. Hill, M. L. Wong, and R. S. Hegde. 1997. A multistep,ATP-dependent pathway for assembly of human immunodeficiency viruscapsids in a cell-free system. J. Cell Biol. 136:567–581.

33. Lingappa, J. R., R. L. Martin, M. L. Wong, D. Ganem, W. J. Welch, and V. R.Lingappa. 1994. A eukaryotic cytosolic chaperonin is associated with a highmolecular weight intermediate in the assembly of hepatitis B virus capsid, amultimeric particle. J. Cell Biol. 125:99–111.

34. Liu, Q., C. Tackney, R. A. Bhat, A. M. Prince, and P. Zhang. 1997. Regulated

processing of hepatitis C virus core protein is linked to subcellular localiza-tion. J. Virol. 71:657–662.

35. Lo, S. Y., M. J. Selby, and J. H. Ou. 1996. Interaction between hepatitis Cvirus core protein and E1 envelope protein. J. Virol. 70:5177–5182.

36. Lohmann, V., F. Korner, A. Dobierzewska, and R. Bartenschlager. 2001.Mutations in hepatitis C virus RNAs conferring cell culture adaptation.J. Virol. 75:1437–1449.

37. Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Barten-schlager. 1999. Replication of subgenomic hepatitis C virus RNAs in ahepatoma cell line. Science 285:110–113.

38. Lu, W., and J. H. Ou. 2002. Phosphorylation of hepatitis C virus core proteinby protein kinase A and protein kinase C. Virology 300:20–30.

39. Ma, H. C., C. H. Ke, T. Y. Hsieh, and S. Y. Lo. 2002. The first hydrophobicdomain of the hepatitis C virus E1 protein is important for interaction withthe capsid protein. J. Gen. Virol. 83:3085–3092.

40. Martin-Serrano, J., and P. D. Bieniasz. 2003. A bipartite late-budding do-main in human immunodeficiency virus type 1. J. Virol. 77:12373–12377.

41. Matsumoto, M., T. Y. Hsieh, N. Zhu, T. VanArsdale, S. B. Hwang, K. S. Jeng,A. E. Gorbalenya, S. Y. Lo, J. H. Ou, C. F. Ware, and M. M. Lai. 1997.Hepatitis C virus core protein interacts with the cytoplasmic tail of lympho-toxin-beta receptor. J. Virol. 71:1301–1309.

42. McLauchlan, J. 2000. Properties of the hepatitis C virus core protein: astructural protein that modulates cellular processes. J. Viral Hepat. 7:2–14.

43. McLauchlan, J., M. K. Lemberg, G. Hope, and B. Martoglio. 2002. In-tramembrane proteolysis promotes trafficking of hepatitis C virus core pro-tein to lipid droplets. EMBO J. 21:3980–3988.

44. Melton, D. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn, and M. R.Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNAhybridization probes from plasmids containing a bacteriophage SP6 pro-moter. Nucleic Acids Res. 12:7035–7056.

45. Mizuno, M., G. Yamada, T. Tanaka, K. Shimotohno, M. Takatani, and T.Tsuji. 1995. Virion-like structures in HeLa G cells transfected with thefull-length sequence of the hepatitis C virus genome. Gastroenterology 109:1933–1940.

46. Moriishi, K., T. Okabayashi, K. Nakai, K. Moriya, K. Koike, S. Murata, T.Chiba, K. Tanaka, R. Suzuki, T. Suzuki, T. Miyamura, and Y. Matsuura.2003. Proteasome activator PA28gamma-dependent nuclear retention anddegradation of hepatitis C virus core protein. J. Virol. 77:10237–10249.

47. Moriya, K., H. Fujie, Y. Shintani, H. Yotsuyanagi, T. Tsutsumi, K. Ishibashi,Y. Matsuura, S. Kimura, T. Miyamura, and K. Koike. 1998. The core proteinof hepatitis C virus induces hepatocellular carcinoma in transgenic mice.Nat. Med. 4:1065–1067.

48. Moriya, K., H. Yotsuyanagi, Y. Shintani, H. Fujie, K. Ishibashi, Y. Mat-suura, T. Miyamura, and K. Koike. 1997. Hepatitis C virus core proteininduces hepatic steatosis in transgenic mice. J. Gen. Virol. 78(Pt 7):1527–1531.

49. Mukhopadhyay, S., P. R. Chipman, E. M. Hong, R. J. Kuhn, and M. G.Rossmann. 2002. In vitro-assembled alphavirus core-like particles maintaina structure similar to that of nucleocapsid cores in mature virus. J. Virol.76:11128–11132.

50. Nolandt, O., V. Kern, H. Muller, E. Pfaff, L. Theilmann, R. Welker, andH. G. Krausslich. 1997. Analysis of hepatitis C virus core protein interactiondomains. J. Gen. Virol. 78:1331–1340.

51. Owsianka, A. M., and A. H. Patel. 1999. Hepatitis C virus core proteininteracts with a human DEAD box protein DDX3. Virology 257:330–340.

52. Perara, E., and V. R. Lingappa. 1985. A former amino terminal signalsequence engineered to an internal location directs translocation of bothflanking protein domains. J. Cell Biol. 101:2292–2301.

53. Pietschmann, T., V. Lohmann, A. Kaul, N. Krieger, G. Rinck, G. Rutter, D.Strand, and R. Bartenschlager. 2002. Persistent and transient replication offull-length hepatitis C virus genomes in cell culture. J. Virol. 76:4008–4021.

54. Poynard, T., V. Ratziu, Y. Benhamou, P. Opolon, P. Cacoub, and P. Bedossa.2000. Natural history of HCV infection. Baillieres Best Pract. Res. Clin.Gastroenterol. 14:211–228.

55. Sacher, R., R. French, and P. Ahlquist. 1988. Hybrid brome mosaic virusRNAs express and are packaged in tobacco mosaic virus coat protein in vivo.Virology 167:15–24.

56. Sandefur, S., R. M. Smith, V. Varthakavi, and P. Spearman. 2000. Mappingand characterization of the N-terminal I domain of human immunodefi-ciency virus type 1 Pr55(Gag). J. Virol. 74:7238–7249.

57. Santolini, E., G. Migliaccio, and N. La Monica. 1994. Biosynthesis andbiochemical properties of the hepatitis C virus core protein. J. Virol. 68:3631–3641.

58. Shih, C. M., C. M. Chen, S. Y. Chen, and Y. H. Lee. 1995. Modulation of thetrans-suppression activity of hepatitis C virus core protein by phosphoryla-tion. J. Virol. 69:1160–1171.

59. Shih, C. M., S. J. Lo, T. Miyamura, S. Y. Chen, and Y. H. Lee. 1993.Suppression of hepatitis B virus expression and replication by hepatitis Cvirus core protein in HuH-7 cells. J. Virol. 67:5823–5832.

60. Smith, A. J., N. Srinivasakumar, M. L. Hammarskjold, and D. Rekosh. 1993.


Requirements for incorporation of Pr160gag-pol from human immunodefi-ciency virus type 1 into virus-like particles. J. Virol. 67:2266–2275.

61. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: gene expression,replication, and evolution. Microbiol. Rev. 58:491–562.

62. Tellinghuisen, T. L., A. E. Hamburger, B. R. Fisher, R. Ostendorp, and R. J.Kuhn. 1999. In vitro assembly of alphavirus cores by using nucleocapsidprotein expressed in Escherichia coli. J. Virol. 73:5309–5319.

63. Wengler, G., U. Boege, H. Bischoff, and K. Wahn. 1982. The core protein ofthe alphavirus Sindbis virus assembles into core-like nucleoproteins with the

viral genome RNA and with other single-stranded nucleic acids in vitro.Virology 118:401–410.

64. You, L. R., C. M. Chen, T. S. Yeh, T. Y. Tsai, R. T. Mai, C. H. Lin, and Y. H.Lee. 1999. Hepatitis C virus core protein interacts with cellular putative RNAhelicase. J. Virol. 73:2841–2853.

65. Zhu, N., A. Khoshnan, R. Schneider, M. Matsumoto, G. Dennert, C. Ware,and M. M. Lai. 1998. Hepatitis C virus core protein binds to the cytoplasmicdomain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J. Virol. 72:3691–3697.


identiﬁcation of residues in the hepatitis c virus core

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