INVESTIGATING HEPADNAVIRAL CAPSID

ENVELOPMENT AND VIRION PRODUCTION

Natalie J. Greco

A dissertation submitted in partial fulfillment of the requirements for

the degree of

Doctor of Philosophy

(Cellular and Molecular Pathology)

at the

UNIVERSITY OF WISCONSIN-MADISON

2015

Date of final oral examination: 12/11/2014

The dissertation is approved by the following members of the Final Oral Committee: Daniel D. Loeb, Professor, Oncology Paul Ahlquist, Professor, Oncology and Molecular Virology Shannon Kenney, Professor, Oncology and Medicine William Sugden, Professor, Oncology Marulasiddappa Suresh, Associate Professor, Pathobiological Sciences

i

Abstract

Hepadnaviruses selectively package capsids containing mature dsDNA genomes

into virions. The research presented in this dissertation provides insight into this poorly

understood aspect of viral replication. Snow goose hepatitis B virus (SGHBV) is the only

known hepadnavirus that packages capsids containing immature ssDNA into virions. I

found that cells replicating SGHBV produce virions containing ssDNA as efficiently as

virions containing dsDNA and that they support high levels of virion production,

compared to DHBV. I determined that SGHBV capsid protein (Cp) and large envelope

protein (L) independently contribute to the ability of SGHBV to produce virions

containing ssDNA with Cp making a larger contribution. Also, I found that L contributes

to the high levels of virion production characteristic of SGHBV. I conferred these

properties onto DHBV by substituting regions of the SGHBV proteins into corresponding

DHBV proteins, allowing me to identify residues within Cp and L that are responsible for

the different properties of these viruses.

I identified two amino acid residues of DHBV Cp that contribute to selective

dsDNA virion production and may interact with the envelope proteins during virion

formation. Additionally, I identified a region of DHBV L that contributes to selective

dsDNA virion production. I found that this same region of L was also responsible for

DHBV’s relatively low levels of virion production. Future studies on the role of these

residues in virion production will broaden our understanding of this aspect of virus

replication.

Finally, I found that HHBV envelope proteins cannot package DHBV or SGHBV

capsids into virions. I used this incompatibility to identify residues of Cp involved in

ii

virion formation. I substituted a small segment of HHBV Cp into DHBV Cp and this

restored the ability of HHBV envelope proteins to package DHBV capsids into virions.

Residues within this segment likely interact with envelope proteins during virion

morphogenesis. Interestingly, this segment contains the residues of Cp responsible for

selective dsDNA virion production. A similar approach can be taken to identify regions

of the envelope proteins involved in capsid packaging and virion production.

iii

Acknowledgements

First and foremost, I would like to thank Dan Loeb. I feel extremely fortunate to

have had Dan Loeb as my dissertation advisor. I am certain that I would not have gotten

as much out of this process, had I done my dissertation research anywhere but at Loeb

University. Dan’s enthusiasm for the research he does is inspiring. Always leading by

example, Dan trained me to think critically and creatively about my work, and the work

of others. He could be “harsh” at times but he was always “fair”. I appreciated this trait in

him because it helped me to identify and work on my weaknesses to become a better

scientist. Dan provided me with just the right balance of freedom to explore what

interested me and guidance to ensure that I accomplished my goals. Most importantly,

he taught me to think thoroughly about every single thing I do. Dan, I am forever

appreciative of the time and effort you put forth to help me become the scientist I am

today. I will miss you and cannot thank you enough for the lessons you taught me in lab

and in life!

I would also like to thank the current members of Loeb University, Karolyn Pionek

and Nuruddin Unchwaniwala, for always being eager to discuss interesting results and

provide feedback on my research. Thank you for making Loeb U such an awesome and

enjoyable place to be and for always being there if I needed anything, in or out of lab!

This has certainly been the best time of my life, in part because I got to come to the lab

and see you two and Dan every day! Thanks for everything, I will miss you both so

much!

I would like to thank Mike Hayes, a former research technician in our lab, for

providing his technical support when I first joined the lab and for his contributions to the

iv

early stages of my dissertation research. Mike was the first to suggest that we start

using SGHBV to study capsid maturation and selective virion production. He made

several of the initial SGHBV plasmids, from which all of the subsequent SGHBV

plasmids were derived. I would also like to thank former graduate students, Thomas

Lentz and Eric Lewellyn, who were senior graduate students when I joined the lab and

were great graduate student role models.

I would like to thank my committee members, Paul Ahlquist, Shannon Kenney,

Bill Sugden and M Suresh, for providing me with valuable feedback and advice at my

committee meetings. I am additionally grateful to Bill for taking time to share his

thoughts on choosing an appropriate post-doctoral research position.

I feel fortunate to have been able to do my dissertation research at McArdle

Laboratory for Cancer Research. I would like to acknowledge the founding fathers (and

mother) of McArdle Laboratory. These scientists deserve credit for shaping the McArdle

community into what it is today. McArdle is, and always has been, a very collaborative

and supportive community of researchers who are deeply invested in developing the

scientific abilities of their graduate students and post-docs. I would like to thank

everyone at McArdle for making this such a great place to work… from the janitors to

the administrative staff to the director of the department. I’d particularly like to mention

the ladies from Lambertville, my Beatle buddy Gaye and our dear friend Jodi, and all of

the CMP and cancer biology graduate students!

I would also like to thank all of my friends and family in Chicago for always

offering their words of encouragement throughout my time in graduate school. I am

grateful to my parents for always making my education a priority. Finally, I would like to

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thank all of the friends that I have made in Madison; in particular Toni, Heather, Ben and

the ladies at the Lakehouse! I will miss you all and thanks for all the good times!

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Table of Contents

Abstract .................................................................................................................i

Acknowledgements ............................................................................................ iii

Table of Contents ............................................................................................... vi

List of Figures .................................................................................................... ix

List of Abbreviations .......................................................................................... xi

Chapter 1. Hepatitis B Virus Background and Introduction ............................1

HBV transmission, disease and treatment ..................................................2

Hepadnaviridae ..........................................................................................5

Genome organization and viral proteins .....................................................9

Viral replication ........................................................................................ 16

Selective production of virions containing mature dsDNA ........................ 24

Chapter 2: Materials and Methods ................................................................... 26

Chapter 3: Snowgoose Hepatitis B Virus (SGHBV) Capsid and

Envelope Proteins Contribute to the Ability of SGHBV to Package

Capsids Containing ssDNA in Virions ............................................................. 35

Abstract .................................................................................................... 36

Importance................................................................................................ 38

Introduction ............................................................................................... 39

Results ..................................................................................................... 43

Rationale ............................................................................................. 43

Characterizing SGHBV virion production ............................................ 43

vii

Cp contributes to SGHBV’s ability to efficiently package capsids

containing ssDNA in virions ........................................................... 45

Residues 74 and 107 of Cp contribute to DHBVs ability to

selectively package capsids containing mature dsDNA

genomes in virions ......................................................................... 47

Residues 74 and 107 of Cp contribute to SGHBVs ability to

efficiently produce virions containing ssDNA ................................. 49

Changing residues 74 and 107 of HHBV Cp does not confer the

ability to produce virions containing ssDNA onto HHBV ................ 50

SGHBV envelope proteins are sufficient to cause high levels of

virion production and the production of virions containing

ssDNA ............................................................................................ 51

A determinant within residues 61 and 120 of SGHBV L

contributes to the high levels of virion production and the

production of virions containing ssDNA characteristic of

SGHBV .......................................................................................... 52

Discussion ................................................................................................ 65

Chapter 4: Identifying Amino Acid Residues of Avihepadnaviral

Capsid and Envelope Proteins That Contribute to the Packaging of

Capsids into Virions .......................................................................................... 73

Abstract .................................................................................................... 74

Introduction ............................................................................................... 75

Results ..................................................................................................... 78

viii

HHBV envelope proteins cannot package SGHBV or DHBV

capsids into virions ........................................................................ 78

A determinant within amino acid residues 69 and 114 of Cp is

involved in virion production ........................................................... 80

Discussion ................................................................................................ 87

Chapter 5: Summary and Future Directions ................................................... 88

References ......................................................................................................... 95

ix

List of Figures

Figure 1.1. Structure of HBV viral particles ...........................................................8

Figure 1.2. Coding organization and viral transcripts .......................................... 12

Figure 1.3. Capsid protein (Cp) structure and capsid assembly .......................... 13

Figure 1.4. DHBV large (L) and small (S) surface protein topologies .................. 15

Figure 1.5. Viral replication strategy .................................................................... 21

Figure 1.6. Genome replication strategy ............................................................. 23

Figure 3.1. SGHBV supports high levels of virion production and efficiently

produces virions containing ssDNA .......................................................... 56

Figure 3.2. SGHBV Cp contributes to the efficient production of virions

containing ssDNA, while SGHBV envelope proteins contribute to the high

levels of virion production, characteristic of SGHBV ........................................... 57

Figure 3.3. Residues 74 and 107 of DHBV Cp contribute to the selective

production of virions containing dsDNA .................................................... 60

Figure 3.4. Residues 74 and 107 of SGHBV Cp contribute to the

production of virions containing ssDNA ............................................................... 61

Figure 3.5. Changing residues 74 and 107 of HHBV Cp is not sufficient to

cause the production of virions containing ssDNA .............................................. 62

Figure 3.6. A determinant within residues 61 and 120 of L contributes to

the selective production of virions containing dsDNA ............................... 63

Figure 3.7. Phylogenetic tree based on capsid protein amino acid

sequence .................................................................................................. 64

x

Figure 4.1. HHBV envelope proteins cannot package SGHBV capsids into

virions. A determinant between amino acid residues 22 and 139 of

Cp contributes to virion production ........................................................... 82

Figure 4.2. HHBV envelope proteins cannot package DHBV capsids into

virions. A determinant between amino acid residues 69 and 114 of

Cp contributes to virion production ........................................................... 84

Figure 4.3. Region of the capsid protein (Cp) found to contribute to virion

production contains the residues of Cp involved in selective production of

virions containing dsDNA .................................................................................... 86

xi

List of Abbreviations

Cp capsid protein

cccDNA covalently closed circular DNA

DHBV duck hepatitis B virus

DL DNA duplex linear DNA

DR1,2 direct repeat 1, 2

ε epsilon – cis-acting sequence

HBeAg hepatitis B virus e-antigen

HBV human hepatitis B virus

HCC hepatocellular carcinoma

HHBV heron hepatitis B virus

HIV human immunodeficiency virus

iRC DNA incomplete relaxed circular DNA

kb kilobase

kDa kilodalton

L large surface/envelope protein

M medium surface/envelope protein

nt(s) nucleotide(s)

ORF open reading frame

P viral reverse transcriptase/polymerase protein

pgRNA pregenomic RNA

RC DNA relaxed circular DNA

S small surface/envelope protein

xii

SGHBV snow goose hepatitis B virus

sgRNA subgenomic RNA

SS DNA single stranded DNA

TP terminal protein domain of P

vDNA virion associated DNA

vRI encapsidated viral replicative intermediate

WHV woodchuck hepatitis B virus

WT wild type

1

1

2

CHAPTER 1 3

4

5

HEPATITIS B VIRUS BACKGROUND AND INTRODUCTION 6

7

2

HBV Transmission, Disease and Treatment 8

As its name implies, human hepatitis B virus (HBV) causes inflammation of the liver. 9

HBV can cause an acute or a chronic infection. During a chronic infection, viral 10

replication persists and the prolonged inflammation and hepatocyte cell death caused 11

by the ongoing infection can lead to severe liver disease, such as liver cirrhosis or 12

hepatocellular carcinoma (HCC). Acute infections are typically resolved naturally without 13

treatment and only cause mild disease. HBV can be transmitted through infected blood 14

and bodily fluids. Globally, the most common route of transmission is from mother to 15

child at birth. It is estimated that 200-500 million individuals are chronically infected 16

worldwide, with around one million people dying each year from diseases associated 17

with chronic infection1–4. Prevalence of HBV infection varies greatly between geographic 18

locations. HBV is most prevalent in Sub-Saharan Africa and is less of a concern in the 19

United States (and other high income countries). It is estimated that in 2005, greater 20

than 8% of the population was chronically infected in Sub-Saharan Africa, while less 21

than 2% of the population was chronically infected in the United States5. 22

Interestingly, whether HBV causes acute versus chronic infection in an individual is 23

largely dependent on the age at which infection occurs. Around 90% of individuals 24

infected as an adult will clear the infection. In stark contrast to this, only 5-10% of those 25

infected at birth or as an infant will clear the infection; the large majority of these 26

individuals will become life-long chronic carriers of the virus and many will die of 27

diseases associated with chronic infection. The risk for chronic HBV infection decreases 28

for children between the ages 1 and 4; 30% of those infected will become chronically 29

3

infected. Hence, the risk of chronicity is inversely related to the age at which infection 30

occurs6–8. 31

Individuals chronically infected with HBV are at an increased risk for developing and 32

ultimately dying from HCC9,10. HCC is the sixth most common form of cancer worldwide 33

and the third most common cause of death by cancer11. It is thought that the 34

inflammatory immune response to infection and high rate of cell turnover contribute to 35

the development and/or maintenance of HCC. However, the mechanism by which HBV 36

contributes is not defined. Because chronic HBV infection is one of the leading causes 37

of HCC, the best way to decrease the incidence of HCC is to prevent new HBV 38

infections through the use of vaccines. 39

A safe and effective vaccine was introduced in the early 1980s which has led to a 40

dramatic decrease in HBV prevalence and a decrease in HCC incidence in many parts 41

of the world5,12. However, because vaccination rates are low in some rural and low 42

income parts of the world chronic HBV infection remains a major world health concern. 43

Further, the vaccine is prophylactic and is ineffective at treating individuals already 44

chronically infected. 45

A way to decrease HBV associated mortality is through the use of anti-viral 46

therapies. There are seven approved antiviral agents to treat chronic infection, with 47

more in preclinical and clinical trials13–15. Current therapies can be divided into two 48

types; 1) nucleos(t)ide analogues (NAs) targeted at the viral DNA polymerase, such as 49

entecavir or tenofovir, and 2) immunomodulatory/antiviral agents, such as interferon 50

(IFN) or pegylated-IFN. However, these therapies can lead to drug resistance16 and can 51

have severe adverse side effects, respectively. Further, while these treatments 52

4

suppress viral replication, none of the current treatment options eliminate the virus. 53

Because of the lack of a cure, there is a great need to develop new therapies that can 54

cure chronic HBV infection. A deeper understanding of the molecular biology underlying 55

the replication of the virus could provide insight into the design and development of 56

therapies targeted at different aspects of replication and increase likelihood of 57

eradicating the virus and the devastating diseases it causes. 58

59

5

Hepadnaviridae 60

HBV is the prototypic member of the family Hepadnaviridae, derived from the words 61

hepatic DNA virus. All share a similar replication strategy and have many characteristics 62

in common: (1) All hepadnaviruses are dsDNA viruses, which replicate their genomes 63

via an RNA intermediate, known as the pregenomic RNA (pgRNA). This is in contrast to 64

retroviruses, such as human immunodeficiency virus (HIV) which is an RNA virus and 65

replicates its genome by integrating into the genome of an infected cell. Because of this 66

difference, HBV has been classified as a para-retrovirus. (2) Genome replication occurs 67

within cytoplasmic capsids and is facilitated by the virally encoded polymerase (P) 68

protein. The P protein has several enzymatic activities, such as RNA- and DNA-69

dependent DNA polymerase activities and RNase H activity, which allow it to reverse 70

transcribe the viral genome. And (3) all hepadnaviruses are enveloped viruses. Several 71

viral envelope proteins and presumably host-derived lipids form the envelope of the 72

virion. Because virions are not produced in the absence of envelope proteins, the 73

envelope proteins are thought to play an active role in virion production17,18. 74

Viruses in the Hepadnaviridae family can be divided into ortho- and avi- 75

hepadnaviruses, found in a variety of mammals and a variety of avian species, 76

respectively. Because of the narrow host range of hepadnaviruses19–22 and the lack of 77

an effective in vivo infection model system for the human virus, non-human 78

hepadnaviruses have been invaluable models and have helped us better understand 79

how hepadnaviruses replicate. Duck hepatitis B virus (DHBV)23 has been widely studied 80

to elucidate the molecular biology of hepadnaviruses both in vitro and in vivo24. This is 81

in large part due to the availability of well-established in vitro and in vivo model systems. 82

6

Primary duck hepatocytes (PDHs), which can be harvested from either the livers of 83

Peking ducks or their embryos, have been widely used because of their availability and 84

because they can be infected in vitro. Transfection of a chicken hepatoma cell line 85

(LMH)25 has also been used extensively to study multiple aspects of replication. Virions 86

produced by LMH cells are infectious in Peking ducks and PDHs, allowing the 87

opportunity to analyze effects of mutating the virus in its natural host; making this 88

transfection system extremely useful. 89

Much of what we know about how hepadnaviruses replicate was first learned 90

through the study of duck hepatitis B virus (DHBV) and later tested in the human virus. 91

Other members of this family which have been useful model systems include 92

woodchuck hepatitis B virus (WHV)26, and of note to my dissertation, snow goose 93

hepatitis B virus (SGHBV)27 and heron hepatitis B virus (HHBV) 28. While all 94

hepadnaviruses are similar, they are not identical in their pathogenesis or in the 95

diseases they cause. Therefore, some are better suited to study certain aspects of the 96

virus than others. For example, because avihepadnaviruses do not cause liver cancer in 97

their natural hosts, DHBV is not a useful model system to study HBV’s role in HCC. 98

Instead, WHV has been used to study how HBV contributes to the development of 99

HCC. 100

Another major difference between ortho- and avi- hepadnaviruses is that 101

orthohepadnaviruses express two additional proteins, the X and the M proteins. The M 102

protein is an envelope protein present in orthohepadnaviral virions. M is thought to be 103

non-essential to virus replication, because knocking down its expression does not 104

hinder viral replication or virion production. The X protein has been shown to interact 105

7

with a large number of cellular proteins, but a consensus on its exact role in viral 106

replication has not been reached. 107

HBV virions are also called Dane particles, named after their discoverer who first 108

visualized virions in the serum of Australian patients. All hepadnaviruses share a similar 109

virion structure (Figure 1.1); Virions have a diameter of 42 nm29 and 45 nm30 for the 110

human and duck hepatitis B viruses respectively. They consist of an outer lipoprotein 111

envelope which surrounds an inner protein shell (known as the nucleocapsid). The 112

virion envelope can be removed by treatment with non-ionic detergents (such as NP40), 113

leaving the capsids intact31. Nucleocapsids from the human and duck hepatitis B virus 114

are both 34 nm in diameter and are made of 240 copies of the capsid protein (Cp). The 115

P protein, which is covalently attached to the 5’ end of the minus-strand of the dsDNA 116

genome32, resides within the nucleocapsid. Nucleocapsids can be treated with SDS and 117

proteases to release the viral genome and remove the P protein from the minus-strand, 118

making isolation of virion associated viral DNA straightforward. 119

There are two forms of the dsDNA genome that can be found within virions. The 120

predominant form is the relaxed circular genome (RC DNA). RC DNA is a partially 121

dsDNA molecule consisting of a full length minus-strand and an incomplete plus-strand. 122

RC DNA is held in a circular conformation through overlapping 5’ cohesive ends. The 123

other dsDNA genome, termed duplex linear DNA (DL DNA), is a linear dsDNA molecule 124

and is much less abundant. These two forms differ in the mechanism by which they are 125

synthesized (Figure 1.6). These two different forms are synthesized via mutually 126

exclusive pathways, which I will describe in more detail later. 127

8

128

Figure 1.1. Structure of HBV viral particles. 129

(A) Left Schematic representation of an avihepadnaviral virion. The lipoprotein envelope of the virion 130

contains the viral large (L) and small (S) surface proteins, depicted in green. Center The envelope can be 131

removed with a mild detergent treatment, leaving the icosahedral capsid intact. The structural protein of 132

the capsid is the capsid/core protein (Cp), which is depicted in red. Right The viral genome can be 133

released from within the nucleocapsid with treatment of SDS and the P protein (depicted in yellow) can be 134

removed from the 5’ end of the minus-strand with a protease treatment. The plus-strand has a short 135

oligoribonucleotide (wavy line) at its 5’ end. (B) Electron micrograph image showing three types of HBV 136

viral particles33

; 1) virions or Dane particle, 2) filamentous sub-viral particles (SVPs) and 3) spherical 137

20nm SVPs. 138

139

9

Genetic Organization and Viral Proteins 140

All hepadnaviruses have a very similar genetic organization. Their genomes are 141

typically 3 to 3.2 kb in length, and code for a small number of proteins. They have 142

overlapping genes and each nucleotide codes for at least one protein. 143

Avihepadnaviruses have three genes, which code for four proteins (Figure 1.2). The P 144

gene codes for the multi-functional 90kDa P protein. P can be divided into several 145

functional domains, which are highly conserved among hepadnaviruses. These 146

domains from N-terminus to C-terminus are the terminal protein, spacer, reverse 147

transcriptase and RNase H. The reverse transcriptase and RNase H domains share 148

sequence and functional homology with other reverse transcriptases. P differs from 149

other reverse transcriptases in that P is required for selective packaging of pgRNA into 150

capsids34,35, and P serves as a protein primer for reverse transcription of the pgRNA. 151

The tyrosine residue at amino acid position 96 of DHBV P supplies the priming hydroxyl 152

group32,36. 153

The C gene codes for the capsid protein (Cp), which is the structural subunit of the 154

nucleocapsid. DHBV Cp is 262 amino acids in length and has a molecular weight of 32 155

kDa. DHBV Cp is larger than its human HBV counterpart (Figure 1.3A), which is only 156

183 amino acids in length and has a molecular weight of 21 kDa. Cp can be functionally 157

divided into two domains; the N-terminal domain and the C-terminal domain. The N-158

terminal domain is often called the assembly domain because it is sufficient for capsid 159

assembly37,38. The C-terminal domain (CTD) is highly basic, is required for pgRNA 160

encapsidation but not capsid assembly39,40 and can act as a nucleic acid chaperone41. 161

Lewellyn and Loeb have shown that the CTD contributes pleiotropically to several steps 162

10

of genome replication42. Cp can be phosphorylated at several arginine-rich regions in 163

the CTD. It is thought that the phosphorylation status of the protein correlates with 164

different stages of the replication cycle37,43–46. 165

The structure of the human HBV capsid has been determined47; however this 166

was done using a C-terminally truncated version of Cp, not full-length Cp. From 3D 167

reconstructions we see that the capsid has protruding spikes studding its surface and 168

has 2 nm pores that are large enough to allow dNTPs to freely pass in and out of the 169

capsid (Figure 1.3B). While high resolution structures of the DHBV capsid are not 170

available, cryo-EM analyses48 and structural models made using different methods 171

37,49,50 support the idea that DHBV capsids have a similar structure. 172

The “preC” portion of the C gene is used to express the e-antigen, which is similar to 173

Cp except that it is N-terminally extended and C-terminally truncated. Similar to HBV e-174

antigen (HBeAg), DHBV e-antigen (DHBeAg) can be found in the serum of infected 175

ducks. The role of e-antigen in HBV infection/replication is unknown. 176

The preS/S gene of avian hepadnaviruses consists of the PreS and S domains and 177

encodes two envelope proteins, the large (L) and the small (S) proteins. Both proteins 178

contain the C-terminal S domain. They differ in that the L protein is N-terminally 179

extended by ~163 amino acids (depending on the virus and isolate) and contains both 180

the PreS and the S domains. L and S are expressed from separate mRNAs which are 181

both transcribed from the PreS/S gene. Both proteins have complex transmembrane 182

topologies, spanning the membrane several times (Figure 1.4). The L protein is known 183

to take on at least two topologies51–55; one in which the PreS domain is cytosolically 184

11

disposed and one in which the PreS domain is disposed within the lumen of the 185

vesicular membrane it resides in. 186

The L protein’s multiple topologies allow it to perform its different functions which 187

require it to be on the interior, as well as exterior of the virion. For example, on the 188

exterior of the virion, the L protein is thought to mediate entry by binding to a cellular 189

receptor19,56–59. When cytosolically disposed, it can interact with nucleocapsids (directly 190

or indirectly) and facilitate the packaging of capsids into virions. 191

Orthohepadnaviruses have four genes which code for six proteins; the X and M 192

proteins in addition to those expressed in avihepadnaviruses. The M protein is an 193

envelope protein and is expressed from its own transcript. The X protein is coded by the 194

X gene and is expressed from its own transcript. 195

196

12

197

Figure 1.2. Coding organization and viral transcripts. 198

(A) DHBV has a 3kb genome. The genome contains overlapping open reading frames (ORFs). The C 199

gene (red) codes for the capsid protein (Cp), the P gene (yellow) codes for the polymerase (P) protein, 200

and the PreS/S gene (green) codes for the large (L) and small (S) surface proteins. (B) The innermost 201

circle represents the cccDNA found in the nucleus. Capped (cap) and poly-adenylated (An) subgenomic 202

RNAs (sgRNAs, depicted in green) and pregenomic RNA (pgRNA, depicted in blue) are transcribed from 203

the cccDNA. Arrows indicate direction of transcription and locations of direct-repeats 1 and 2 (DR1 and 204

DR2) are indicated with grey boxes. (C) Representation of viral transcripts. The pgRNA (blue) codes for 205

the Cp and P proteins, but also serves as the template for genome replication. Lengths of the pgRNA and 206

sgRNAs are noted on the right, DR1 and DR2 are indicated by grey boxes, the two copies of epsilon on 207

the pgRNA are indicated by a small ε and the terminal redundancy on the pgRNA is indicated by an R. 208

13

209

210

211

212

14

Figure1.3. Capsid protein (Cp) structure and capsid assembly. 213

(A) Linear representations of the DHBV and HBV capsid proteins (image is taken directly from49

). The 214

assembly and C-terminal domains (CTD) are labelled above the representations. The regions which form 215

the capsid spikes are represented by light grey, the proposed “insertion domain” within DHBV Cp is 216

indicated by the hashed pattern and the morphogeneic regions are indicated by a thick black line. (B) 3D 217

reconstruction of the crystal structure of the HBV capsid. The surface of the capsid is studded with spikes 218

and is fairly porous. (C) Ribbon representations of a HBV Cp monomer and dimer. The CTD is not 219

depicted here; these structures represent Cp which is truncated at amino acid 144. (D) Residues found to 220

be involved in HBV virion formation shown on a ribbon representation of a Cp dimer. Residues found to 221

be involved in virion formation are depicted with green spheres and are labelled in white60

. 222

223

15

224

Figure 1.4. DHBV large (L) and small (S) surface protein topologies. 225

Top The vesicular membrane in which the envelope proteins reside is depicted by two black lines, with 226

the luminal and cytosoloic (or the virion exterior or interior respectively) are indicated. L takes on at least 227

two known topologies, with the PreS region being disposed on either side of the membrane. A region of L 228

thought to be involved in virion morphogenesis/capsid interactions is indicated by a blue box. DHBV L is 229

also known to be phosphorylated at the serine at amino acid 118 (S118)61,62

, which is indicated by a 230

yellow starburst. This phosphorylation is not thought to play a role in assembly or infectivity. L is also 231

myristoylated at its N-terminus63

, this modification is indicated by a purple X shape. Bottom Model of 232

selective packaging of capsids containing dsDNA into virions; only capsids containing dsDNA can interact 233

with the envelope proteins to be enveloped and packaged into a virion. 234

235

16

Viral Replication 236

Hepadnaviruses preferentially replicate in hepatocytes. They use a combination of 237

host and viral proteins to ultimately cause these cells to release infectious virions non-238

cytolytically (Figure 1.5). Upon entry, the viral nucleocapsid is trafficked to the nucleus 239

(Figure 1.5A), where its genome is deposited into the nucleus and modified to form a 240

super-coiled, covalently closed circular dsDNA (cccDNA) (Figure 1.5B). This process 241

involves several modifications, including completion of plus-strand synthesis, removal of 242

the P protein from the minus-strand DNA, removal of the RNA used to prime plus-strand 243

synthesis and ligation of the DNA strands. cccDNA plays an essential role in sustaining 244

chronic infection and viral persistence. Because of this, host and viral proteins involved 245

in the synthesis of cccDNA are attractive drug targets. Unfortunately, how HBV 246

synthesizes and maintains cccDNA in the nucleus is not well understood. 247

cccDNA is transcribed by cellular RNA polymerase II for the synthesis of pgRNA and 248

subgenomic RNAs (sgRNAs) (Figure 1.5C). The various transcripts are initiated from 249

different promoters but all use the same (and only) poly-adenylation site within the 250

genome (Figure 1.2B). The subgenomic RNAs, which are generated from differential 251

transcription of a single ORF, code for the L and S envelope proteins. L and S are 252

thought to be co-translationally inserted into the membrane of a cellular secretory 253

vesicle. The pgRNA serves as the replication template and also codes for the Cp and P 254

proteins (Figure 1.2C). 255

Cp and P proteins are expressed once pgRNA is exported from the nucleus. As Cp 256

accumulates in the cytoplasm, capsid proteins dimerize to form T-shaped structures in 257

which two alpha helices from each Cp monomer bundle together to form (what will 258

17

eventually be) the capsid spikes (Figure 1.3C). These dimers go on to form trimers of 259

dimers, which are thought to quickly coalesce to form an icosahedral capsid 260

structure64,65. Capsids can form with either a T=3 (90 dimers) or T=4 (120 dimers) 261

symmetry. Of the capsids formed from full-length Cp, approximately ~90% of the 262

capsids will have a T=4 symmetry. The T=3 symmetry becomes more favored as the 263

CTD is progressively truncated66. The role of T=3 capsids, if any, in viral replication is 264

not defined. 265

The P protein interacts with an encapsidation signal within the pgRNA, known as 266

epsilon, and this ribonucleoprotein complex becomes enclosed within the capsid, 267

forming the nucleocapsid through a process known as pgRNA encapsidation (Figure 268

1.5D). Capsids can self-assemble and it is unclear whether P and the pgRNA are 269

encapsidated before or after capsid assembly (for a review see67). 270

Genome replication is a rather complicated process, involving template switches that 271

are facilitated by a number of cis-acting sequences throughout the genome. Of 272

particular importance are the 11-12 nt long complementary sequences at either end of 273

the genome (Figure 1.2C), termed direct-repeat 1 and 2 (DR1 and DR2). Using several 274

residues in epsilon at the 5’ end of the pgRNA, P synthesizes four nucleotides of the 275

minus-strand. Serving as a protein primer in this process, P supplies the priming 276

hydroxyl from a tyrosine residue in its TP domain and becomes covalently attached to 277

the minus-strand36,68,69. P then switches templates to a complementary site near the 3’ 278

end of the pgRNA template, which overlaps with 4nt of DR170–72 (Figure 1.6 A). Here, P 279

continues to synthesize the minus-strand with its RNA-dependent DNA polymerase 280

activity as it degrades the pgRNA with its RNase H activity (Figure 1.6 B). This results in 281

18

a terminally-redundant minus-strand. P leaves a small RNA fragment (of ~18-19 nt) at 282

the 3’ end of the minus-strand which it uses as a primer to subsequently synthesize the 283

plus-strand of the genome73. This capped oligoribonucleotide is present on the final 284

dsDNA molecule found in virions (Figure 1.1A). 285

Typically, this RNA primer is transferred from DR1 to a partially complementary site 286

termed DR2, near the 5’ end of the minus strand in a process called primer 287

translocation and plus-strand synthesis begins73 (Figure 1.6 C). The plus-strand is 288

extended to the 5’ end of its template and the third and final template switch occurs. The 289

3’ end of the plus-strand to anneal to the 3’ end of the minus-strand in a process called 290

circularization (Figure 1.6 D)74; extension of the plus-strand from this site leads to the 291

formation of RC DNA. In a small number of instances, the primer does not translocate to 292

DR2 and plus-strand synthesis is initiated from DR1. This is called in situ priming 293

(Figure 1.6 E) and leads to the formation of the linear DL DNA form of the genome70. 294

Once the plus-strand is synthesized, capsids can be trafficked to the nucleus to 295

deposit the genome where the partially dsDNA genome is converted to cccDNA; 296

increasing the reservoir of cccDNA molecules in the nucleus (Figure 1.5 E). 297

Alternatively, the capsids can acquire an envelope in a process known as capsid 298

envelopment. While this step of replication is not well understood, it is thought that 299

capsids acquire an envelope by budding into the membrane of a secretory vesicle 300

(possibly a post-ER pre-golgi vesicular structure75) containing the trans-membrane 301

envelope proteins. After which, these enveloped nucleocapsids are guided through a 302

constitutive secretion pathway, non-cytolytically producing virions. Unfortunately, the 303

viral and host components involved and the mechanisms underlying this process are 304

19

not understood. Recent efforts have led to a slightly better understanding of the host 305

proteins and processes that are utilized or manipulated during virion formation, for 306

example cellular components involved in autophagy or vesicular/endosomal 307

trafficking76–83 have been proposed to contribute to virion production. However, much 308

more work needs to be done to define a canonical virion formation pathway. 309

Fortunately, more is known about the role that the viral capsid and envelope proteins 310

play in virion morphogenesis. Regions of the capsid 60,84,85 and envelope 18,86–89 proteins 311

involved in virion morphogenesis have been studied extensively (more so for HBV than 312

DHBV, for reviews see17,90). Both L and S are required for virion formation; in the 313

absence of the envelope proteins capsids are not released from the cell within a lipid 314

shell. This suggests the envelope proteins play an active role in coordinating capsid 315

envelopment and virion formation. It is hypothesized that the PreS region of the L 316

protein acts as a matrix protein, interacting with the capsid prior to capsid budding and 317

guiding it to be packaged into virions. A short sequence of the PreS domain of L has 318

been shown to be involved in virion formation87,88. For DHBV, a region between amino-319

acids 117 and 135 of DHBV L has been shown to contribute to virion morphogenesis, 320

because mutating this region decreased virion production while not affecting capsid 321

assembly. The location of this region in the proposed topological structure of DHBV L 322

(Figure 1.4) would support the idea that this region can interact with mature cytoplasmic 323

nucleocapsids; consistent with the hypothesis that L acts as a matrix protein to 324

envelope and package capsids into virions. As for Cp, several residues within the 325

assembly domain at the base of the capsid spikes are thought to play a role in virion 326

morphogenesis and potentially envelope protein interactions; when these residues were 327

20

changed in HBV, Cp capsid assembly and genome replication occurred normally but 328

virions were not produced (Figure 1.3 D). Because of this, it has been proposed that the 329

envelope interacting site is at the base of the capsid spikes. However, given the capsid 330

structure and physical proximity to the envelope, some propose the envelope-interacting 331

site is at the tip of the capsid spikes. It is possible that the envelope-capsid interaction is 332

a two-step process; for example, the first interaction may occur at the tip of the capsid 333

spikes and a second interaction occurs at the base of the spikes. 334

Interestingly, in addition to producing virions, infected cells also produce what are 335

known as sub-viral particles (SVPs) (Figures 1.1B and 1.5H). These particles are either 336

filamentous particles of various lengths or spherical; both particles are 20nm in 337

diameter. Similar to virions, SVPs contain the L and S envelope proteins. However, 338

SVPs differ from virions in that they do not contain a nucleocapsid and are therefore 339

non-infectious. DHBV virions and SVPs contain both the L and S proteins, with the S 340

protein being around four times more abundant in both54,91,92. 341

It is estimated that SVPs are produced at 1,000-10,000 fold higher levels than 342

virions. The role, if any, these particles play in viral replication is not completely 343

understood. Interestingly, SVPs enhance infection at low multiplicities of infection 93. It 344

has been proposed that these particles act as a decoy for the immune system, allowing 345

virions which would otherwise be cleared by the immune response to avoid surveillance. 346

Another idea is that these particles may bind cellular receptors of uninfected cells, 347

stimulating signaling pathways which make these cells more permissive for infection by 348

HBV. 349

21

350

Figure 1.5. Viral replication strategy. 351

A) Upon entry, the nucleocapsid deposits its genome into the nucleus, through a process that is not 352

well defined. 353

B) This genome is modified to form a covalently closed circular DNA (cccDNA) molecule. 354

C) From cccDNA, subgenomic RNAs (sgRNAs) and pregenomic RNA (pgRNA) are transcribed by 355

cellular RNA pol II. 356

D) Polymerase (P) and capsid (Cp) proteins are expressed from pgRNA and begin to accumulate in 357

the cytoplasm. At some point, P interacts with a secondary structure on the pgRNA and this 358

ribonucleoprotein complex becomes encapsidated. This process is known as pgRNA 359

encapsidation. 360

E) Within the cytoplasmic capsids, the P protein reverse transcribes the pgRNA, forming the minus-361

strand of the genome and subsequently synthesizes the plus-stand. This gives rise to a dsDNA 362

22

viral genome. P mediated reverse transcription is discussed in further detail within the text and a 363

schematic representation of the process is depicted in Figure 1.6. 364

F) Capsids containing mature dsDNA genomes can be trafficked back to the nucleus to deposit their 365

genomes; increasing the copy number of cccDNA within the nucleus. This process is often called 366

cccDNA amplification. The L protein of DHBV and HBV have been shown to play a role in 367

regulating cccDNA amplification94,95

. 368

G) The sgRNAs code for the large (L) and small (S) envelope proteins, which are co-translationally 369

inserted into the membrane of a vesicular structure (some suggest a post-ER pre-Golgi vesicle). 370

As the envelope proteins accumulate in this membrane, they are thought to interact with each 371

other and with mature cytoplasmic nucleocapsids to facilitate capsid envelopment and the 372

packaging of capisds into virions. Support for the second function comes from the fact that both L 373

and S are required for virion formation. 374

H) The L and S proteins can also form particles which lack a nucleocapsid (and are therefore non-375

infectious), known as sub-viral particles (SVPs). Once L and S have accumulated to a certain 376

level and ratio, they are thought to aggregate and bud inward towards the lumen of the vesicle in 377

which they reside. These SVPs are thought to be released from the cell via a constitutive 378

secretion pathway, but the mechanisms underlying their formation are not defined. 379

380

23

381

Figure 1.6. Genome replication strategy. 382

The two forms of the dsDNA genome, relaxed circular (RC DNA) and duplex linear (DL DNA) are formed 383

via mutually exclusive pathways. Both pathways begin with P interacting with the pgRNA packaging 384

signal, epsilon, and synthesizing ~4nt of the minus-strand. (A) P, along with the nascent minus-strand 385

switches templates to the copy of DR1 at the 3’ end of the pgRNA. (B) P elongates the minus-strand, as it 386

degrades the pgRNA. P leaves a small RNA fragment at the 3’ end of the minus-strand which it uses as a 387

primer for plus-strand synthesis. (C) The RNA primer switches templates and anneals to DR2 and plus-388

strand synthesis starts. (D) The final step in the synthesis of RC DNA is the last template switch, 389

facilitated by the terminal redundancies within the minus-strand template; the nascent plus-strand anneals 390

to the 3’ end of the minus-strand where plus-strand synthesis resumes. The final RC DNA molecule is 391

held in a circular conformation by its 5’ cohesive ends. (E) If the primer does not translocate from DR1 at 392

the 3’ end of the minus-strand to DR2 and minus-strand is elongated from this location, a DL DNA 393

molecule is formed. This is referred to as in situ priming and occurs at a low frequency. 394

24

Selective production of virions containing mature dsDNA 395

It has long been appreciated that hepadnaviruses selectively produce virions 396

containing mature dsDNA genomes. Capsids containing pgRNA are not packaged into 397

virions, while capsids containing ssDNA have been found to be packaged at a very low 398

efficiency96–99. Even in the absence of capsids containing dsDNA, capsids containing 399

pgRNA or ssDNA are not packaged into virions. Because of this, it is thought that 400

capsids containing mature dsDNA genomes differ from all other capsids in their ability to 401

interact with envelopment machinery and be packaged into a virion. 402

It is thought that the capsid serves as a link between genome replication and capsid 403

envelopment, relaying information about the completeness of genome replication to the 404

capsid surface. One model is that the synthesis of dsDNA triggers a physical change to 405

occur on the exterior of the capsid. This change is often referred to as the “capsid 406

maturation signal” or “capsid packaging signal” and renders the capsid competent for 407

packaging into virions and may facilitate interactions required for virion formation to 408

occur. In this way, the virus is able to prevent capsids containing incomplete/immature 409

genomes from being enveloped and packaged into virions. 410

Mutating HBV Cp at residue 97 causes the formation of virions containing ssDNA100–411

103. I similarly found that mutating a single residue of DHBV Cp causes DHBV to 412

produce virions containing ssDNA and present this work in chapter 3. Interestingly, 413

mutations in the HBV L protein can offset this secretion of virions containing immature 414

genomes, restoring preferential production of HBV virions containing mature dsDNA 415

genomes104. This implies that the envelope proteins are also involved in discriminating 416

between capsids containing mature and immature genomes. Consistent with these 417

25

findings, I present evidence which supports the idea that the envelope proteins, 418

specifically a small contiguous region of the DHBV PreS region of the L protein, 419

contribute to the ability of the virus to discriminate between capsids containing mature 420

and immature genomes. My findings suggest that L actively selects capsids which 421

contain mature dsDNA genomes for envelopment and selective packaging into virions 422

(Figure 1.4) and challenge the long-standing model that selective production of virions 423

containing dsDNA genomes is coded solely by the capsid. 424

425

26

426

427

CHAPTER 2 428

429

430

MATERIALS AND METHODS 431

432

27

Molecular Clones 433

DHBV plasmids: All DHBV molecular clones are derived from DHBV3105. The WT 434

DHBV plasmid, pD1.5G, has been described previously and contains 1.5 tandem copies 435

of DHBV3 DNA106. The DHBV L and S protein donor, DHBVEnv+, is a monomer of 436

DHBV3 in the vector pSP65. It has been previously described as pD3-SP65107. Only the 437

L and S envelope proteins are expressed from DHBVEnv+. The DHBV plasmid deficient 438

in Cp expression, DHBVpgRNA+P+Env+, expresses WT DHBV pgRNA, P, L and S proteins, 439

it contains a 4-nucleotide deletion at the NsiI site within the C gene, such that functional 440

Cp is not expressed. The DHBV plasmid deficient in envelope protein expression, 441

DHBVpgRNA+P+C+, expresses WT DHBV pgRNA, P and Cp. It contains a T1327A change, 442

which introduces a premature stop codon in the S gene. 443

The DHBV plasmid that expresses only Cp, DHBVC+, contains a deletion of nt 444

424 that creates a premature stop codon in the P gene and a deletion from nts 2549-445

2580 that inactivates the encapsidation signal. It also contains the inactivating mutation 446

in the S gene described above. 447

All DHBV Cp variants were derived from DHBVC+. Overlap extension PCR was 448

performed to create SG 74-107 D Cp. The single amino acid changes were introduced 449

using overlap extension PCR and oligonucleotide-directed mutagenesis108. PCR 450

fragments were inserted into DHBVC+. At residue 74, leucine was changed to an 451

isoleucine to create L74I DHBV Cp. At residue 87, glutamine was changed to a serine 452

to create Q87S DHBV Cp. At residue 107, histidine was changed to glutamic acid to 453

create H107E DHBV Cp. 454

28

SGHBV plasmids: All SGHBV molecular clones are derived from a plasmid 455

expressing SGHBV1-15 27. The WT SGHBV plasmid contains 1.3 tandem copies of 456

SGHBV1-15 DNA inserted into the PstI site of pBS-. The SGHBV L and S protein donor, 457

SGHBVEnv+, is a monomer of SGHBV1-15 inserted in the pBS- vector. Only L and S 458

envelope proteins are expressed from SGHBVEnv+. 459

The SGHBV plasmid deficient in Cp expression, SGHBVpgRNA+P+Env+, expresses 460

WT SGHBV pgRNA, P, L and S proteins. It contains a G2854T change within the C 461

gene, such that functional Cp is not expressed. The SGHBV plasmid deficient in 462

envelope protein expression, SGHBVpgRNA+P+C+, expresses WT SGHBV pgRNA, P and 463

Cp. It contains a TC to AA change at nt 1300, which introduces a premature stop codon 464

in the S gene. 465

The SGHBV plasmid which expresses only Cp, SGHBVC+, contains an insertion 466

at nt 426 that creates a premature stop codon in the P gene and a deletion from nts 467

2552-2582 that inactivates the encapsidation signal. It also contains the inactivating 468

mutations in the S gene described above. 469

All SGHBV Cp variants were derived from SGHBV1-15 Cp donor plasmid, 470

SGHBVC+. The amino acid changes were introduced into the SGHBV Cp gene using 471

overlap extension PCR and oligonucleotide-directed mutagenesis108. At residue 74, 472

isoleucine was changed to leucine to create I74L SGHBV Cp. At residue 107, glutamic 473

acid was changed to histidine to create E107H SGHBV Cp. These two changes were 474

combined to create the double mutant 74L 107H SGHBV Cp. 475

All L protein variants were derived from DHBVEnv+ and SGHBVEnv+. The plasmid 476

PreS-S D-SG L expresses a WT SGHBV S protein and a chimeric L protein consisting 477

29

of a DHBV PreS domain and an SGHBV S domain. PreS-S D-SG L was made by 478

inserting a KpnI-AvrII fragment from WT SGHBV into DHBVEnv+. The reciprocal plasmid 479

PreS-S SG-D L expresses a WT DHBV S protein and a chimeric L protein consisting of 480

an SGHBV PreS domain and a DHBV S domain. PreS-S SG-D L was made by inserting 481

a KpnI-NcoI fragment from WT DHBV into SGHBVEnv+. Overlap extension PCR was 482

performed to make chimeric envelope proteins SG 1-118 D L and SG 61-120 D L. PCR 483

fragments were inserted into DHBVEnv+. Both plasmids express WT DHBV proteins and 484

chimeric L proteins. The chimeric L proteins are primarily DHBV but contain SGHBV 485

sequence from amino acids 1-118 or 61-120, respectively. 486

HHBV plasmids. All molecular clones of HHBV are derived from a plasmid 487

expressing HHBV428; this plasmid is also referred to as 413-2. 413-2 contains 1.4 488

tandem copies of HHBV4 DNA inserted into an EcoRI site on the vector pIBI21106. The 489

HHBV L and S protein donor, HHBVEnv+, is a monomer of HHBV4 inserted in the pIBI21 490

vector. Only L and S envelope proteins are expressed from HHBVEnv+. 491

Details describing the HHBV Cp protein donor plasmid, HHBVC+, have been 492

previously described109. The HHBV plasmid deficient in Cp expression, 493

HHBVpgRNA+P+Env+, expresses WT HHBV pgRNA, P, L and S proteins, it contains a 494

frameshift mutation resulting from a 4nt insertion at the HindIII site at nucleotide 38, 495

such that functional Cp is not expressed. The HHBV plasmid deficient in L and 496

expression, HHBVpgRNA+P+C+, has been previously described and referred to as 497

pHSS1106. The plasmid expresses WT HHBV pgRNA, P, and Cp proteins, it contains a 498

mutation resulting in a premature stop codon in the S gene, such that functional L and S 499

are not expressed. 500

30

All HHBV Cp variants were derived from the HHBV4 Cp donor plasmid, HHBVC+. 501

PCR was performed using make chimeric Cp variants H 22-139 SG Cp and H 69-114 D 502

Cp. PCR fragments were inserted into HHBVC+. The H 22-139 SG Cp plasmid 503

expresses a chimeric Cp that is primarily HHBV, except for at 25 amino acid residues 504

between residues 22 and 139, as well as at the C-terminal residue. The H 69-114 D Cp 505

plasmid expresses a chimeric Cp that is primarily DHBV Cp, except for at eleven 506

residues within residues 69 and 114. 507

Overlap extension PCR was performed to create SG 74-107 D Cp. The three 508

HHBV Cp variants, L74I HHBV Cp, N107E HHBV Cp and 74I 107E HHBV Cp. The 509

amino acid changes were introduced into the HHBV Cp gene using overlap extension 510

PCR and oligonucleotide-directed mutagenesis108. At residue 74, leucine was changed 511

to isoleucine to create L74I HHBV Cp. At residue 107, asparagine was changed to 512

glutamic acid to create N107E HHBV Cp. These two changes were combined to create 513

the double mutant 74I 107E HHBV Cp. 514

Cell culture and transfection 515

Chicken hepatoma cell line, LMH25,110, was used in all transfections. Cells were 516

cultured and transfected as previously described111 with minor adjustments. Briefly, cells 517

were grown in Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (Gibco) and 518

were supplemented to a final concentration of 5% fetal bovine serum and 519

penicillin/streptomycin. Cells were seeded onto 60 mm dishes 24 hours prior to 520

transfection. Plasmid DNA (10.5 ug total) was transfected into cells at 70-80% 521

confluence. In co-transfection experiments, the ratio of C protein donor plasmids to C 522

deficient plasmids was 1:1. Each transfection included 0.5 ug of a plasmid expressing 523

31

green fluorescent protein to estimate transfection efficiency. Transfections were 524

performed using the calcium phosphate method112. Media containing the calcium-525

phosphate precipitate was left on the cells for 16-18 hours. Cells were washed with 526

HBS-EGTA (2mM HEPES, 150mM NaCl, 0.5mM EGTA, pH 7.45) and fresh media was 527

replaced. Counting this time-point as 0 hours, media was changed and discarded at 24 528

hours. Subsequently, media was collected at 72 and 96 hours and pooled for virion 529

DNA isolation. After 96 hours, cells were washed with HBS-EGTA (2mM HEPES, 530

150mM NaCl, 0.5mM EGTA, pH 7.45) and stored at -700C overnight for isolation of viral 531

replicative intermediates from cytoplasmic capsids. 532

Isolation of viral replicative intermediates from cytoplasmic capsids 533

Isolation of viral replicative intermediates from cytoplasmic capsids was 534

performed as previously described113. Briefly, cells were lysed with a solution of 50mM 535

Tris pH 8.0, 1 mM EDTA, 0.2% NP-40. Nuclei were pelleted by centrifugation at 15,000 536

x g for 5 minutes at 40C and discarded. Supernatants were brought to 2mM CaCl2 and 537

treated with 44 units of micrococcal nuclease to degrade transfected plasmid DNA. After 538

1.5 hours, EDTA was added to a final concentration of 10mM to inactivate micrococcal 539

nuclease activity. Viral replicative intermediates (vRIs) were released from cytoplasmic 540

capsids and the P protein was removed from the viral DNA by the addition of Pronase to 541

a final concentration of 0.4 mg/ml (Roche) and SDS to a final concentration of 0.4%. 542

Following a 2-hour incubation at 370C, vRIs were extracted with phenol/chloroform, 543

ethanol precipitated, resuspended in TE (10mM Tris, 0.1mM EDTA. pH 8.0) and treated 544

with 2 ug of RNase A. 545

546

32

Isolation of virion DNA from LMH culture media 547

Virions were isolated as described114 with minor modifications. Briefly, pooled 548

culture media was centrifuged at 1,200 x g for 15 minutes to remove dead cells and 549

debris. To precipitate the viral particles, PEG 8000 and NaCl were added to a final 550

concentration of 10% (w/v) and 0.5M, respectively. Samples were incubated overnight 551

at 40C on a rocking platform and virions were pelleted via centrifugation at 3,200 x g for 552

15 minutes. Virions were resuspended in 400 ul of Leibovitz’s L-15 Medium (Invitrogen) 553

and buffered by the addition of Tris (pH 8.0) to a final concentration of 75mM. To 554

remove free capsids, Pronase (Roche) was added to a final concentration of 0.4 mg/ml 555

and incubated at 370C for 1-1.5 hours. This treatment was sufficient to degrade 556

unenveloped capsids, but not capsids within virions114. We demonstrated this was true 557

for SGHBV and DHBV. Viral DNA released from unenveloped capsids was degraded by 558

adding 44 units of micrococcal nuclease and CaCl2 to a final concentration of 2mM. 559

After one hour, EDTA was added to a final concentration of 10mM to inactivate 560

micrococcal nuclease activity. Virion DNA (vDNA) was released from virions by the 561

addition of SDS to a final concentration of 0.4%. The P protein was removed from the 562

viral DNA by adding Pronase (Roche) to a final concentration of 0.4 mg/ml and 563

incubating samples for 2 hours at 370C. Virion DNA was extracted with 564

phenol/chloroform, ethanol precipitated, resuspended in TE (10mM Tris, 0.1mM EDTA. 565

pH 8.0) and treated with 2 ug of RNase A for 30 minutes at 370C. 566

567

33

Southern blot analysis of viral nucleic acid 568

The method used for Southern blotting has been previously described42,115 with 569

minor alterations. Briefly, vRI and vDNA were electrophoresed through a 1.25% 570

agarose gel in Tris-borate-EDTA buffer (90mM Tris-borate, 2.5mM EDTA, pH 8.5). A 571

0.8% agarose gel was used when the packagable pgRNA in the sample being analyzed 572

was derived from HHBV. DNA was transferred to a Hybond-N membrane (Amersham) 573

and UV cross-linked to the membrane. Membranes were incubated in Church 574

hybridization solution (5mM EDTA, 1% BSA, 0.25 M Na2PO4, 7% SDS) for 15 minutes 575

and then probed overnight at 420C. The probe used was comprised of 20 576

oligonucleotides which were end-labeled with [γ-P32]ATP (Perkin-Elmer) using T4 poly-577

nucleotide kinase (New England Biolabs). All oligonucleotides used for probing are 578

complementary to the DHBV and SGHBV minus-strand. Membranes were washed in 579

Church wash solution (1mM EDTA, 20mM Na2PO4, 1% SDS). The membrane was 580

exposed in a phosphorimaging cassette, which was scanned with a Typhoon 8610 581

Variable Mode Imager (Molecular Dynamics). vRI and vDNA were quantitated using 582

ImageQuant 5.2 software (GE Healthcare). Mass of full-length minus-strand DNA was 583

determined by comparison to known masses of a linear double-stranded fragment of the 584

DHBV genome. 585

Statistical Analyses 586

All statistical analyses were done using the program MStat5.5 (provided by Norman 587

Drinkwater, UW-Madison <http://mcardle.wisc.edu/mstat/>). Statistical comparisons 588

34

between samples were made using the Wilcoxon rank sum test (two-sided). All samples 589

had n ≥ 6. We considered P < 0.05 to be statistically significant. 590

591

35

592

593

CHAPTER 3 594

595

596

SNOWGOOSE HEPATITIS B VIRUS (SGHBV) CAPSID AND 597

ENVELOPE PROTEINS CONTRIBUTE TO THE ABILITY OF 598

SGHBV TO PACKAGE CAPSIDS CONTAINING ssDNA IN 599

VIRIONS 600

601

With the exception of Figures 3.5 – 3.7, the data from this chapter has been published 602

in the Journal of Virology 603

(Greco, N., Hayes, M.H., and D.D. Loeb. 2014. J. Virol. 88(18):10705-13) 604

605

The data from figures 3.5 – 3.7 will be expanded upon and submitted as a separate 606

manuscript to the Journal of Virology. 607

36

Abstract 608

Hepadnaviruses selectively package capsids containing mature dsDNA genomes in 609

virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that 610

packages capsids containing ssDNA in virions. We found that cells replicating SGHBV 611

produce virions containing ssDNA as efficiently as virions containing mature dsDNA. 612

We determined that SGHBV capsid protein (Cp) and large envelope protein (L) 613

independently contribute to the production of virions containing ssDNA; with Cp making 614

a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp 615

contribute to this feature of SGHBV. When we changed these residues in DHBV Cp to 616

their SGHBV counterparts, capsids containing immature ssDNA were packaged in 617

virions. Interestingly, when we changed these residues in another avihepadnavirus, 618

heron hepatitis B virus (HHBV), capsids containing immature ssDNA were still not 619

packaged into virions. These results suggest that residues 74 and 107 contribute to the 620

appearance of the “capsid packaging signal” on the surface of capsids and interact with 621

the envelope proteins during virion formation, but that other residues of Cp and/or the 622

envelope proteins contribute as well. We also identified that a determinant within amino 623

acids 61-120 of SGHBV L contributes to its ability to produce virions containing ssDNA. 624

When we substituted this region of SGHBV L into DHBV L, capsids containing ssDNA 625

were packaged into virions. This result uncovers a new function of L and indicates that a 626

determinant between residues 61 and 120 of DHBV L contributes to its ability to 627

preferentially produce virions containing ssDNA. This, conversely, suggests that this 628

region of SGHBV L contributes to its ability to produce virions containing ssDNA. We 629

also found that cells replicating SGHBV package a larger fraction of the total RC DNA 630

37

they synthesize in virions compared to DHBV. We found that SGHBV L (and specifically 631

a determinant between amino acids 61-120) is responsible for this property of SGHBV. 632

Determining if the ability of SGHBV L to cause the formation of virions containing 633

ssDNA is related to its ability to package a large fraction of the total RC DNA they 634

synthesize in virions or if these two properties are mechanistically distinct will provide 635

insights into virion morphogenesis. 636

637

38

Importance 638

Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and 639

immature genomes. However, only capsids containing mature dsDNA genomes are 640

packaged into virions. A mechanistic understanding of this phenomenon, which is 641

currently lacking, is critical to understanding the process of hepadnaviral virion 642

morphogenesis. In this study, we determined that the L protein (and specifically a small 643

region of the PreS of L) contributes to the ability of hepadnaviruses to selectively 644

produce virions containing mature dsDNA genomes. Our finding sheds new light on the 645

mechanisms underlying virion morphogenesis and challenges the dogma that “capsid 646

maturation”, and therefore the capsid protein (Cp), is solely responsible for the selective 647

production of virions containing mature dsDNA genomes. Further, we identified amino 648

acid residues of Cp that contribute to its ability to cause the selective production of 649

virions containing mature dsDNA genomes. Future studies on the role of these residues 650

in selective packaging of capsids containing dsDNA will broaden our understanding of 651

this poorly understood aspect of virus replication. 652

653

39

Introduction 654

Human Hepatitis B Virus (HBV) is the prototypic member of the Hepadnaviridae 655

family. Related hepadnaviruses, such as duck hepatitis B virus (DHBV)23, have been 656

invaluable in understanding HBV replication24. All hepadnaviruses are enveloped 657

viruses and preferentially replicate in hepatocytes116. All hepadnaviruses replicate their 658

genomes via reverse transcription of an RNA intermediate, the pregenomic RNA 659

(pgRNA)117. In this study, we used related avian hepadnaviruses (AHBVs), DHBV and 660

snow goose hepatitis B virus (SGHBV), to investigate capsid envelopment and virion 661

formation. 662

Avian hepadnavirus virions are composed of four viral proteins, the polymerase 663

protein (P), capsid protein (Cp) and the large (L) and small (S) envelope proteins. The 664

virion envelope consists of the L and S envelope proteins and host-cell derived 665

phospholipids118. Beneath the envelope, is the icosahedral nucleocapsid. The capsid is 666

35 nm in diameter and consists of 120 dimeric subunits of Cp. The P protein, which is 667

covalently attached to the ~3kb dsDNA genome, resides within the capsid. 668

Upon entry into a cell, the capsid delivers the viral genome to the nucleus, where 669

it is converted into covalently closed circular DNA (cccDNA). Viral RNAs, including 670

pgRNA, are transcribed from the cccDNA and exported from the nucleus. The pgRNA 671

codes for the Cp and P proteins and also serves as the template for genome replication. 672

During capsid assembly, the P protein binds to the encapsidation signal, called 673

epsilon34,35,107. Cp dimers are thought to polymerize around this ribonucleoprotein 674

complex, forming the capsid37. 675

40

Genome replication takes place within the cytoplasmic capsid and is facilitated by 676

the P protein117. Initially, P reverse transcribes the pgRNA into the minus-strand of the 677

DNA genome as it degrades the pgRNA. P then synthesizes plus-strand DNA, giving 678

rise to two forms of the dsDNA genome; relaxed circular (RC DNA) and duplex linear 679

(DL DNA). RC DNA is the predominant dsDNA form, while DL DNA is less abundant. 680

During an infection, cytoplasmic capsids contain a spectrum of replicative intermediates, 681

ranging from pgRNA to partially synthesized DNA genomes to completely synthesized 682

mature dsDNA genomes. Capsids containing mature dsDNA have two known fates; 683

they can be enveloped and secreted as a virion or they can deliver the viral DNA to the 684

nucleus to amplify and then maintain the copy number of cccDNA119. 685

The L and S proteins are coded by a single open reading frame, which consists 686

of a PreS domain and an S domain. The two proteins share the S domain. They differ in 687

that the L protein is N-terminally extended because it contains the PreS domain. The L 688

and S envelope proteins are both required for virion formation18. The envelope proteins 689

are thought to oligomerize, forming a 3D surface which interacts with cytoplasmic 690

capsids to drive capsid envelopment and virion formation. The envelope proteins are 691

localized in the membrane of a post-ER pre-Golgi secretory vesicle75. It is here where 692

the envelope proteins are hypothesized to interact with the cytoplasmic capsids during 693

virion formation, triggering budding into a secretory vesicle to acquire an envelope. 694

Both L and S have complex trans-membrane topologies. In addition, the L protein 695

is known to have multiple distinct conformations. Initially after synthesis, the PreS region 696

of the L protein is cytosolically disposed, but subsequently the PreS region of a subset 697

of the L proteins translocate across the membrane during virion formation, becoming 698

41

exposed on the exterior of the virion120. The L protein plays a role in virus entry and 699

virion formation; the multiple topologies of L allow it to perform these different functions. 700

Because cytoplasmic capsids contain pgRNA, partially synthesized DNA 701

genomes and mature dsDNA and virions only contain mature dsDNA genomes, it has 702

been hypothesized that the surface of capsids containing mature genomes differs from 703

those containing immature genomes. This difference is referred to as the “maturation” or 704

“capsid packaging signal”117. Capsids that have acquired the “capsid packaging signal” 705

are thought to productively interact with the envelope proteins to ultimately be packaged 706

in virions. Capsids containing ssDNA or pgRNA are thought to lack the “capsid 707

packaging signal”, rendering them incompetent for the interactions required for virion 708

formation. In this way hepadnaviruses selectively produce virions containing dsDNA. 709

Several studies have shown that capsids containing ssDNA or pgRNA are not packaged 710

in virions121, even in the absence of capsids containing dsDNA, supporting this 711

hypothesis96–99. Further, alternative models suggesting that mature capsids are 712

preferentially packaged resulting from an intrinsically higher affinity for interactions with 713

the envelopment machinery or due to kinetics of genome replication have already been 714

excluded through the use of a synchronized secretion system96. 715

Unlike all other hepadnaviruses characterized to date, SGHBV produces virions 716

containing ssDNA27. This property of SGHBV is surprising given its high phylogenetic 717

similarity to DHBV. Our goal was to understand how SGHBV produces virions 718

containing ssDNA, as this would provide insight into how other hepadnaviruses 719

selectively produce virions containing dsDNA. We first characterized the production of 720

virions containing mature dsDNA and immature ssDNA genomes for SGHBV and 721

42

DHBV. To do this, we measured RC DNA and ssDNA in cytoplasmic capsids and 722

extracellular virions. We found that SGHBV packages a larger fraction of the total RC 723

DNA it synthesizes in virions compared to DHBV. Further, we found that cells 724

expressing SGHBV produce virions containing immature ssDNA as efficiently as virions 725

containing mature dsDNA genomes. 726

Next, we determined which SGHBV proteins were responsible for these features 727

of SGHBV. We found that SGHBV Cp and L independently contribute to the production 728

of SGHBV virions containing ssDNA. However, Cp had a larger contribution. We 729

genetically mapped the amino acids of SGHBV Cp that contribute to the production of 730

virions containing ssDNA to residues 74 and 107. Further, we show residues 74 and 731

107 of the DHBV Cp contribute to the ability of DHBV to selectively produce virions 732

containing dsDNA. These residues are likely involved in capsid maturation and could be 733

part of a 3D surface on the exterior of the capsid that interacts with the envelope 734

proteins during capsid envelopment. We found that a determinant within amino acid 735

residues 61 and 120 of SGHBV L contributes to the production of virions containing 736

ssDNA; within this region, SGHBV and DHBV L differ at only 7 amino acid residues. In 737

addition, we found that a determinant within this same region of SGHBV L is 738

responsible for its ability to package a large fraction of the capsids containing RC DNA 739

into virions. It will be interesting to determine if the ability of SGHBV L to cause the 740

production of virions containing ssDNA is a function of its ability to package a large 741

fraction of the capsids containing RC DNA into virions or if these two properties are 742

unrelated. This will provide insight into how SGHBV L contributes to the packaging of 743

capsids containing immature ssDNA in virions. 744

43

Results 745

Rationale 746

It has long been appreciated that hepadnaviruses selectively package capsids 747

containing mature dsDNA in virions. SGHBV is unique in that it produces virions 748

containing dsDNA and ssDNA 27. By studying SGHBV virion production alongside the 749

well-characterized DHBV, we hoped to gain a better understanding of the mechanism 750

by which hepadnaviruses selectively produce virions containing mature dsDNA 751

genomes. Our goal was to confer the ability to produce virions containing ssDNA onto 752

DHBV and, conversely, confer the ability to selectively produce virions containing 753

dsDNA onto SGHBV through genetic complementation. In this way we could identify 754

which viral components were responsible for the respective behaviors of these two 755

viruses. To do this, we first analyzed the production of virions containing dsDNA and 756

ssDNA for DHBV and SGHBV. 757

Characterizing SGHBV virion production 758

LMH cells were transfected with a plasmid expressing SGHBV or DHBV. 759

Cytoplasmic capsid DNA and virion DNA were isolated and Southern blotting was 760

performed. The three major forms of vRIs (RC, DL and SS) were detected in 761

cytoplasmic DHBV capsids (Figure 3.1, lane 3). When DNA was isolated from DHBV 762

virions and analyzed similarly only RC DNA and DL DNA were detected (Figure 3.1, 763

lane 4 ). This result illustrates that DHBV capsids containing mature dsDNA genomes 764

are selectively packaged in virions. As previously described for SGHBV27, all three 765

major forms of the vRIs (RC, DL and SS) were detected in cytoplasmic capsids and 766

44

extracellular virions. This result illustrates that SGHBV produce virions containing 767

immature ssDNA (Figure 3.1, lanes 1 and 2). 768

We wanted to know if SGHBV produced virions containing ssDNA and dsDNA 769

equally well or preferentially produced one type of virion over the other. To this end, we 770

measured the levels of RC DNA and ssDNA in cytoplasmic capsids and in extracellular 771

virions. To quantitatively describe dsDNA virion production, we divided the amount of 772

RC DNA packaged in virions by the sum of RC DNA found in cytoplasmic capsids and 773

extracellular virions and multiplied this value by 100 to obtain a percentage, as shown 774

below: 775

% dsDNA packaged = (RC DNAvirions / (RC DNAcapsids + RC DNAvirions)) x 100 776

For SGHBV, this value was 58 ± 20%, and for DHBV it was 14 ± 3% (Figure 3.1). This 777

result indicated that a larger fraction of the cytoplasmic capsids containing dsDNA were 778

packaged in virions from cells expressing SGHBV compared to DHBV. 779

In the same way, we quantified ssDNA virion production by dividing the amount 780

of ssDNA packaged in virions by the sum of ssDNA found in both cytoplasmic capsids 781

and extracellular virions. We divided ssDNA virion production by RC DNA virion 782

production, to obtain a ratio of ssDNA production to RC DNA production, as shown 783

below: 784

𝑠𝑠𝐷𝑁𝐴 𝑝𝑎𝑐𝑘𝑎𝑔𝑒𝑑𝑑𝑠𝐷𝑁𝐴 𝑝𝑎𝑐𝑘𝑎𝑔𝑒𝑑 =

𝑠𝑠𝐷𝑁𝐴𝑣𝑖𝑟𝑖𝑜𝑛𝑠

𝑠𝑠𝐷𝑁𝐴𝑐𝑎𝑝𝑠𝑖𝑑𝑠 + 𝑠𝑠𝐷𝑁𝐴𝑣𝑖𝑟𝑖𝑜𝑛𝑠

𝑅𝐶 𝐷𝑁𝐴𝑣𝑖𝑟𝑖𝑜𝑛𝑠

𝑅𝐶 𝐷𝑁𝐴𝑐𝑎𝑝𝑠𝑖𝑑𝑠 + 𝑅𝐶 𝐷𝑁𝐴𝑣𝑖𝑟𝑖𝑜𝑛𝑠

785

45

For SGHBV this value was 1.01 ± 0.16, while for DHBV this value was indistinguishable 786

from zero, 0.05 ± 0.09 (Figure 3.1). This result means that SGHBV produces virions 787

containing immature ssDNA as efficiently as virions containing mature dsDNA, 788

indicating SGHBV was indiscriminate in selecting capsids containing dsDNA and 789

ssDNA for envelopment and virion formation. 790

Cp contributes to SGHBV’s ability to efficiently package capsids containing 791

ssDNA in virions 792

Given the capsid maturation hypothesis, we predicted that SGHBV Cp would be 793

sufficient to cause the production of virions containing immature ssDNA. To test this 794

prediction, we co-expressed DHBV P, pgRNA and envelope proteins with SGHBV Cp in 795

LMH cells. We did this by complementing a DHBV plasmid deficient in expressing Cp, 796

DHBVpgRNA+P+Env+, with a SGHBV Cp donor plasmid SGHBVC+. We measured the levels 797

of RC DNA and ssDNA in both cytoplasmic capsids and extracellular virions. We saw 798

no defect in cytoplasmic DNA synthesis or virion production, which allowed us to 799

measure relative ssDNA virion production. We found that relative ssDNA virion 800

production was 0.88 ± 0.11 (Figure 3.2A, lanes 3 and 4). This value was similar to the 801

SGHBV comparison; 0.99 ± 0.14 (Figure 3.2A, lanes 5 and 6). This result indicated that 802

SGHBV Cp contributes to the efficient production of virions containing ssDNA, 803

characteristic of SGHBV. 804

In addition, we found that % dsDNA virion production was low, 26 ± 14%, and 805

similar to the DHBV comparison which was 21 ± 10% (Figure 3.2A, lanes 1-4). This 806

result indicated that, despite its contribution to the efficient production of virions 807

46

containing ssDNA, SGHBV Cp does not contribute to SGHBV’s ability to package a 808

large fraction of the capsids containing RC DNA into virions. 809

To corroborate the above findings, we did complementary analyses in which we 810

co-expressed SGHBV P, pgRNA and Cp with the DHBV envelope proteins. We 811

predicted that we would see low % dsDNA virion production and high levels of relative 812

ssDNA virion production. To test this prediction, we co-transfected SGHBVpgRNA+P+C+ 813

with DHBVEnv+ and measured RC DNA and ssDNA in both cytoplasmic capsids and 814

extracellular virions. We saw no defect in cytoplasmic DNA synthesis or virion 815

production, which allowed us to measure relative ssDNA virion production. We found 816

that relative ssDNA virion production was high, 0.94 ± 0.17, which was similar to the 817

SGHBV comparison, 0.99 ± 0.32 (Figure 3.2B, lanes 5-8). Further, we found that % 818

dsDNA virion production was low, 12 ± 6% (Figure 3.2B, lanes 7 and 8), and was similar 819

to the DHBV comparison, 12 ± 6% (Figure 3.2B, lanes 1 and 2). 820

Taken together, we conclude that SGHBV Cp contributes to SGHBV’s ability to 821

produce virions containing ssDNA as efficiently as virions containing RC DNA, but not 822

its ability to package a large fraction of the capsids containing RC DNA into virions. 823

Because this was true regardless of the origin of P and pgRNA, we were able to 824

summarize these findings in Figure 3.2C. To better understand how SGHBV Cp 825

contributes to the production of virions containing ssDNA, we identified residues of 826

SGHBV Cp responsible for this property of SGHBV Cp, as well as residues of DHBV Cp 827

responsible for its ability to selectively package capsids containing mature dsDNA 828

genomes in virions. 829

47

Residues 74 and 107 of Cp contribute to DHBVs ability to selectively package 830

capsids containing mature dsDNA genomes in virions 831

After establishing that SGHBV Cp contributes to SGHBV’s ability to efficiently 832

package capsids containing ssDNA in virions, we wanted to identify the amino acid 833

residue(s) responsible for this characteristic. Because DHBV Cp and SGHBV Cp differ 834

at only 14 amino acid residues, we reasoned that one, or a few, of these different 835

residues were responsible for the behavior of the respective capsid proteins. According 836

to this logic, we should be able to change the behavior of DHBV Cp from selectively 837

packaging capsids containing mature dsDNA genomes in virions to packaging capsids 838

containing immature ssDNA in virions. We should also be able to change the behavior 839

of SGHBV Cp from packaging capsids containing immature ssDNA in virions to 840

selectively packaging capsids containing mature dsDNA genomes in virions. To identify 841

which residues of Cp were contributing to the respective behaviors of these viruses, we 842

made several chimeric SGHBV-DHBV capsid proteins, which we co-expressed with 843

DHBV P, pgRNA and envelope proteins. Of interest is a chimeric Cp in which we 844

substituted a small region, between residues 74-107, of SGHBV Cp into the DHBV Cp. 845

We called this variant SG 74-107 D Cp and it differs from DHBV Cp at only four 846

positions; 74, 83, 87 and 107 (Figure 3.3A). 847

When DHBV pgRNA, P and envelope proteins were co-expressed with SG 74-848

107 D Cp, we saw that cytoplasmic DNA synthesis and dsDNA virion production were 849

similar to DHBV. More importantly, relative ssDNA virion production was 0.80 ± 0.10 850

(Figure 3.3B, lanes 1 and 2). This value was not different, statistically, than when we 851

48

co-expressed DHBV pgRNA, P and envelope proteins with WT SGHBV Cp, where 852

relative ssDNA virion production was 0.88 ± 0.11 (Figure 3.2A, lanes 3 and 4). 853

Using a published alignment122 of the Cp amino acid sequences of ten other 854

avian hepadnaviruses known to selectively produce virions containing dsDNA and the 855

DHBV3 sequence (which was not included in the published alignment), we identified 856

three residues within this region, 74, 87 and 107, unique to SGHBV Cp. We changed 857

DHBV Cp to SGHBV individually at these three residues and named these proteins; 858

L74I DHBV Cp, Q87S DHBV Cp and H107E DHBV Cp (Figure 3.3A). As before, 859

DHBVpgRNA+P+Env+ and one of the three DHBV Cp variants were co-expressed. 860

Cytoplasmic capsid and virion DNA were isolated and analyzed by Southern blotting. In 861

all three cases, cytoplasmic DNA synthesis and dsDNA virion production were similar to 862

DHBV, which allowed us to evaluate relative ssDNA virion production. 863

When DHBV pgRNA, P and envelope proteins were co-expressed with L74I 864

DHBV Cp (Figure 3.3B, lanes 3 and 4) or H107E DHBV Cp (Figure 3.3B, lanes 7 and 8) 865

relative ssDNA virion production was 0.79 ± 0.22 and 0.34 ± 0.14, respectively. 866

However, when DHBV pgRNA, P and envelope proteins were co-expressed with Q87S 867

DHBV Cp, relative ssDNA virion production was essentially zero, 0.04 ± 0.09 (Figure 868

3.3B, lanes 5 and 6). While changing DHBV Cp to glutamic acid at residue 107, H107E 869

DHBV Cp, caused a significant increase in relative ssDNA virion production when 870

compared to the DHBV comparison, L74I DHBV Cp was the only variant which caused 871

production of ssDNA containing virions to the same relative level as WT SGHBV Cp 872

(Figure 3.2A, lanes 3 and 4). These findings indicate residue 74 plays an important role 873

in coupling genome maturation to capsid envelopment. Because it has been proposed 874

49

that capsid maturation is linked to a change on the exterior of the capsid123, structural or 875

biochemical comparisons of WT DHBV Cp and L74I DHBV Cp capsids could provide 876

insight into the nature of the “capsid packaging signal”. 877

Residues 74 and 107 of Cp contribute to SGHBVs ability to efficiently produce 878

virions containing ssDNA 879

Given the above result, we predicted that changing residues 74 and/or 107 of 880

SGHBV Cp to DHBV would convert SGHBV Cp from a producer of virions containing 881

immature ssDNA to a selective producer of virions containing mature dsDNA genomes. 882

To this end, residues 74 and 107 of SGHBV Cp were changed individually and in 883

combination; I74L SGHBV Cp, E107H SGHBV Cp and 74L 107H SGHBV Cp (Figure 884

3.4A). DHBVpgRNA+P+Env+ and each of the three SGHBV Cp variants were co-expressed, 885

viral DNA was isolated from cytoplasmic capsids and extracellular virions and were 886

analyzed via Southern blotting. We saw that cytoplasmic DNA synthesis and dsDNA 887

virion production were similar to DHBV, which allowed us to evaluate relative ssDNA 888

virion production. 889

When 74 or 107 were changed individually, relative ssDNA virion production was 890

0.26 ± 0.19 and 0.67 ± 0.30, respectively (Figure 3.4B, lanes 1-4). There was not a 891

complete loss of ssDNA virion production in either case. In fact, changing residue 107 892

had almost no effect on the ability of SGHBV Cp to produce virions containing ssDNA. 893

However, when both residues were changed, 74L 107H SGHBV Cp, relative ssDNA 894

virion production was indistinguishable from zero; 0.10 ± 0.09 (Figure 3.4B, lanes 5 and 895

50

6). These results illustrate that residues 74 and 107 of Cp contribute to 896

immature/selective production of virions. 897

Changing residues 74 and 107 of HHBV Cp does not confer the ability to produce 898

virions containing ssDNA onto HHBV 899

To extend these studies, we attempted to confer the ability to produce virions 900

containing ssDNA onto another avihepadnavirus, heron hepatitis B virus (HHBV). We 901

predicted that changing residues 74 and 107 of HHBV Cp to SGHBV would be sufficient 902

to cause HHBV to produce virions containing ssDNA, as it was for DHBV. To test this 903

prediction, we changed these residues in HHBV Cp to SGHBV and made three 904

expression plasmids; L74I H Cp, N107E H Cp and 74L 107E H Cp (Figure 3.5A). We 905

co-transfected LMH cells with HHBVpgRNA+P+Env+, which expresses pgRNA, P, L and S, 906

and a plasmid expressing HHBV Cp or one of the three HHBV Cp variants. Again, 907

cytoplasmic capsid and virion DNA were isolated and analyzed by Southern blotting. In 908

all three cases, cytoplasmic DNA synthesis and dsDNA virion production were similar to 909

HHBV, which allowed us to evaluate relative ssDNA virion production. Contrary to our 910

prediction, ssDNA was not detected in virions (Figure 3.5B). This result indicated that 911

changing residues 74 and 107 in HHBV Cp to SGHBV is not sufficient to confer the 912

ability to produce virions containing ssDNA onto HHBV, as it was for DHBV. DHBV is 913

the most closely related avihepadnavirus to SGHBV, while HHBV is the most distantly 914

related to SGHBV. Because of this, we hypothesize that additional residues in HHBV 915

Cp (or other proteins) need to be changed to convert HHBV from a preferential producer 916

of virions containing dsDNA to a producer of virions containing ssDNA. 917

51

SGHBV envelope proteins are sufficient to cause a large fraction of the capsids 918

containing RC DNA to be packaged into virions and the production of virions 919

containing ssDNA 920

We found that SGHBV packages a larger fraction of the capsids containing RC 921

DNA into virions compared to DHBV (Figure 3.1). We determined that the envelope 922

proteins are responsible for the ability of SGHBV to package a large fraction of the 923

capsids containing RC DNA into virions. When we supplied SGHBV envelope proteins 924

to an envelope-protein deficient DHBV we saw an increase in dsDNA virion production 925

when compared to the DHBV; these values were 45 ± 8% and 12 ± 6%, respectively 926

(Figure 3.2B, lanes 1-4). Conversely, when we supplied DHBV envelope proteins to an 927

envelope-protein deficient SGHBV we saw a decrease in % dsDNA virion production 928

when compared to the SGHBV comparison; these values were 12 ± 6% and 45 ± 20%, 929

respectively (Figure 3.2B, lanes 5-8). We conclude that SGHBV envelope proteins are 930

solely responsible for the ability of SGHBV to package a large fraction of the capsids 931

containing RC DNA into virions. Similarly, DHBV envelope proteins are responsible for 932

the ability of DHBV to package a small fraction of the capsids containing RC DNA into 933

virions. Unexpectedly, we found SGHBV envelope proteins were also sufficient to cause 934

production of virions containing ssDNA; relative ssDNA virion production was 0.61 ± 935

0.20 (Figure 3.2B, lanes 3 and 4). However, this was not as high as the value found 936

when SGHBV Cp was supplied to Cp deficient DHBV, 0.88 ± 0.11 (Figure 3.2A, lanes 3 937

and 4). 938

To corroborate these findings, we performed complementary analyses in which 939

we co-expressed SGHBV P, pgRNA and envelope proteins with DHBV Cp. Because 940

52

SGHBV envelope proteins were present and SGHBV Cp was absent, we expected that 941

virion production would be high and relative ssDNA virion production would be lower 942

than the SGHBV comparison. As expected, % dsDNA virion production was high, 53 ± 943

1% (Figure 3.2A, lanes 7 and 8). This value was similar to the SGHBV comparison 55 ± 944

5% (Figure 3.2A, lanes 5 and 6), supporting the idea that SGHBV’s ability to package a 945

large fraction of the capsids containing RC DNA into virions is a function of its envelope 946

proteins. Further, relative ssDNA virion production was 0.62 ± 0.13 (Figure 3.2A, lanes 947

7 and 8). This value was lower than the SGHBV comparison which was 0.99 ± 0.14 948

(Figure 3.2A, lanes 5 and 6), but higher than the DHBV comparison which was 0.07 ± 949

0.13 (Figure 3.2A, lanes 1 and 2). These results were consistent with our earlier finding 950

that SGHBV envelope proteins contribute to the production of virions containing ssDNA. 951

Taken together, we conclude that SGHBV envelope proteins are sufficient to cause 952

packaging of a large fraction of the capsids containing RC DNA into virions, and also 953

contribute to the production of virions containing ssDNA; as summarized in Figure 3.2C. 954

A determinant within residues 61 and 120 of SGHBV L contributes to the ability of 955

SGHBV to package a large fraction of the capsids containing RC DNA into virions 956

and to produce virions containing ssDNA 957

After establishing that SGHBV envelope proteins contribute to SGHBV’s ability to 958

package capsids containing ssDNA in virions and package a large fraction of the 959

capsids containing RC DNA into virions, we wanted to identify the amino acid residue(s) 960

responsible for these characteristic. It is possible these two traits could map to the same 961

or different residues. As we did with Cp, we reasoned that one, or a few, of the residues 962

which are different in SGHBV and DHBV envelope proteins were responsible for the 963

53

behavior of the respective envelope proteins. According to this logic, we should be able 964

to change the behavior of DHBV envelope proteins from selectively packaging capsids 965

containing mature dsDNA genomes in virions to packaging capsids containing immature 966

ssDNA in virions. Further, we should also be able to change the behavior of SGHBV 967

envelope proteins from packaging capsids containing immature ssDNA in virions to 968

selectively packaging capsids containing mature dsDNA genomes in virions. 969

The S proteins (and the S domain of the L protein) of SGHBV and DHBV are 970

very similar (differing at only 6 amino acid residues), while the PreS domains of their L 971

proteins are less conserved (differing at 24 amino acid residues). Because of this fact, 972

we hypothesized the determinant responsible for the respective behaviors of the 973

SGHBV and DHBV envelope proteins would be within the PreS domain. To test this, we 974

made two chimeric envelope protein constructs in which we swapped the PreS domains 975

of SGHBV and DHBV. The first, PreS-S D-SG L, expresses a WT SGHBV S protein and 976

a chimeric L protein containing a DHBV PreS domain and an SGHBV S domain. The 977

second construct, PreS-S SG-D L, expresses a WT DHBV S protein and a chimeric L 978

protein containing an SGHBV PreS domain and a DHBV S domain. We co-expressed 979

DHBV P, pgRNA and Cp with one of the two chimeric envelope protein constructs and 980

analyzed their ability to package capsids containing RC DNA into virions and to produce 981

virions containing ssDNA. We found that PreS-S D-SG L did not produce virions 982

containing ssDNA and only packaged 24 ± 11% of the RC DNA they synthesize into 983

virions, similar to DHBV envelope proteins. We also found that PreS-S SG-D L behaved 984

similar to the SGHBV envelope proteins; it packaged 42 ± 17% of the RC DNA it 985

synthesized in virions and had a relative ssDNA virion production of 0.54 ± 0.19. This 986

54

allowed us to conclude that a determinant within the PreS region of L contribute to the 987

respective behaviors of these two proteins. 988

We next wanted to identify which residues in the PreS region of L contribute to 989

the respective behaviors of these viruses. To do this, we analyzed several chimeric 990

SGHBV-DHBV L protein constructs, which we co-expressed with DHBV P, pgRNA, Cp 991

and S. Of interest are two chimeric L proteins in which we substituted a small region, 992

between residues 1 and 118 or 61 and 120, of SGHBV L into DHBV L. We called these 993

variants SG 1-118 D L and SG 61-120 D L, respectively. SG 61-120 D L differs from 994

DHBV L at only seven positions; 61, 67, 76, 79, 87, 118 and 120. 995

When DHBV pgRNA, P, Cp and S proteins were co-expressed with SG 1-118 D 996

L or SG 61-120 D L, we saw normal cytoplasmic DNA synthesis, allowing us to evaluate 997

the production of virions. We found that % dsDNA virion production was high for both 998

SG 1-118 D L and SG 61-120 D L; % dsDNA virion production was 58 ± 17% and 41± 999

21%, respectively (Figure 3.6, lanes 5-8). These values were similar to the value seen 1000

when we co-expressed DHBV pgRNA, P, and Cp proteins with WT SGHBV L and S, 1001

where 55 ± 5% of the RC DNA synthesized was packaged into virions (Figure 3.2A, 1002

lanes 5 and 6); indicating that a determinant between residues 61 and 120 of SGHBV L 1003

contributes to its ability to package a large fraction of the capsids containing RC DNA 1004

into virions. 1005

When we evaluated the ability of these L variants to produce virions containing 1006

ssDNA, we found that relative ssDNA virion production was 0.65 ± 0.17 and 0.54 ± 0.19 1007

for SG 1-118 D L and SG 61-120 D L, respectively (Figure 3.6, lanes 5-8). These 1008

55

values were not different, statistically, than when we co-expressed DHBV pgRNA, P, 1009

and Cp proteins with WT SGHBV L and S, where relative ssDNA virion production was 1010

0.61 ± 0.20 (Figure 3.2B, lanes 3 and 4). This result suggests that a determinant(s) 1011

between residues 61 and 120 of SGHBV L contributes to its ability to package capsids 1012

containing ssDNA into virions. 1013

56

1014

FIGURE 3.1. SGHBV packages a large fraction of capsids containing RC DNA 1015

into virions and efficiently produces virions containing ssDNA. 1016

Southern blot analysis of viral DNA isolated from LMH cell cultures transfected with a plasmid 1017

expressing SGHBV or DHBV. Below are the mean values and standard deviations for dsDNA virion 1018

production (% dsDNA pkgd) and relative ssDNA virion production (ssDNA pkgd/dsDNA pkgd). Mean 1019

values represent analysis from at least six independent transfections of each virus; RC = relaxed 1020

circular DNA, DL = duplex linear DNA, SS = single-stranded DNA, C = cytoplasmic capsid DNA, V = 1021

virion DNA. 1022

1023

57

FIGURE 3.2. SGHBV Cp contributes to the efficient production of virions 1024

containing ssDNA, while SGHBV envelope proteins contribute to the ability of 1025

SGHBV to package a large fraction of the capsids containing RC DNA into virions, 1026

characteristic of SGHBV. 1027

58

1028

FIGURE 3.2. SGHBV Cp contributes to the efficient production of virions 1029

containing ssDNA, while SGHBV envelope proteins contribute to the ability of 1030

SGHBV to package a large fraction of capsids containing RC DNA into virions. 1031

59

Southern blot analysis. (A.) A plasmid expressing DHBV pgRNA, P, L and S was co-1032

transfected with a plasmid expressing DHBV Cp (lanes 1 and 2) for a DHBV 1033

comparison or SGHBV Cp (lanes 3 and 4). A plasmid expressing SGHBV pgRNA, P, 1034

L and S was co-transfected with a plasmid expressing SGHBV Cp (lanes 5 and 6) for 1035

an SGHBV comparison or DHBV Cp (lanes 7 and 8). (B.) A plasmid expressing DHBV 1036

pgRNA, P and Cp was co-expressed with a plasmid expressing DHBV L and S (lanes 1037

1 and 2) for a DHBV comparison or a plasmid expressing SGHBV L and S (lanes 3 1038

and 4). A plasmid expressing SGHBV pgRNA, P and Cp was co-transfected with a 1039

plasmid expressing SGHBV L and S (lanes 5 and 6) for an SGHBV comparison or a 1040

plasmid expressing DHBV L and S (lanes 7 and 8). Below are the mean values and 1041

standard deviations for dsDNA virion production (% dsDNA pkgd) and relative ssDNA 1042

virion production (ssDNA pkgd/dsDNA pkgd). Mean values represent analysis from at 1043

least six independent transfections of each virus. (C.) Tables summarizing the effects 1044

of Cp and envelope proteins on dsDNA virion production and relative ssDNA virion 1045

production; RC = relaxed circular DNA, DL = duplex linear DNA, SS = single-stranded 1046

DNA, C = cytoplasmic capsid DNA, V = virion DNA. 1047

1048

60

1049

FIGURE 3.3. Residues 74 and 107 of DHBV Cp contribute to the selective 1050

production of virions containing dsDNA. 1051

(A.) Sequences of the region between amino acid residues 70 and 110 of DHBV Cp and the four DHBV 1052

Cp variants analyzed; SG 74-107 D Cp, L74I DHBV Cp, Q87S DHBV Cp and H107E DHBV Cp. (B.) 1053

Southern blot analysis. A plasmid expressing DHBV pgRNA, P, L and S was co-transfected with a Cp 1054

donor plasmid expressing one of the four DHBV Cp variants. Below are the mean values and standard 1055

deviations for relative ssDNA virion production (ssDNA pkgd/dsDNA pkgd). Mean values represent 1056

analysis from at least six independent transfections of each variant; RC = relaxed circular DNA, DL = 1057

duplex linear DNA, SS = single-stranded DNA, C = cytoplasmic capsid DNA, V = virion DNA. 1058

1059

61

1060

FIGURE 3.4. Residues 74 and 107 of SGHBV Cp contribute to the production of 1061

virions containing ssDNA. 1062

(A.) Sequences of the region between amino acid residues 70 and 110 of SGHBV Cp and SGHBV Cp 1063

variants analyzed; I74L SGHBV Cp, E107H SGHBV Cp and 74L 107H SGHBV Cp. (B.) Southern blot 1064

analysis. A plasmid expressing DHBV pgRNA, P, L and S was co-transfected with a Cp donor plasmid 1065

expressing one of the three SGHBV Cp variants. Below are the mean values and standard deviations 1066

for relative ssDNA virion production (ssDNA pkgd/dsDNA pkgd). Mean values represent analysis from 1067

at least six independent transfections of each variant; RC = relaxed circular DNA, DL = duplex linear 1068

DNA, SS = single-stranded DNA, C = cytoplasmic capsid DNA, V = virion DNA. 1069

1070

62

1071

FIGURE 3.5. Changing residues 74 and 107 of HHBV Cp is not sufficient to 1072

cause the production of virions containing ssDNA. 1073

(A.) Sequences of the region between amino acid residues 70 and 110 of HHBV Cp and HHBV Cp 1074

variants analyzed; L74I HHBV Cp, N107E HHBV Cp and 74L 107E HHBV Cp. (B.) Southern blot 1075

analysis. A plasmid expressing HHBV pgRNA, P, L and S was co-transfected with a Cp donor plasmid 1076

expressing one of the three HHBV Cp variants. 1077

1078

63

1079

1080

FIGURE 3.6. A determinant within residues 61 and 120 of L contributes to the 1081

selective production of virions containing dsDNA. 1082

(A.) Southern blot analysis. A plasmid expressing DHBV pgRNA, P and Cp was co-transfected with an 1083

L and S donor plasmid expressing DHBV S and one of the four L protein variants. Below are the mean 1084

values and standard deviations for relative ssDNA virion production (ssDNA pkgd/dsDNA pkgd). Mean 1085

values represent analysis from at least six independent transfections of each variant; RC = relaxed 1086

circular DNA, DL = duplex linear DNA, SS = single-stranded DNA, C = cytoplasmic capsid DNA, V = 1087

virion DNA. 1088

1089

64

1090

FIGURE 3.7. Phylogenetic tree based on capsid protein amino acid sequence. 1091

Evolutionary relationships of avian capsid proteins. The evolutionary history was inferred using the 1092

Neighbor-Joining method124

. The optimal tree with the sum of branch length = 0.45985976 is shown. The 1093

tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used 1094

to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction 1095

method125

and are in the units of the number of amino acid substitutions per site. The analysis involved 26 1096

amino acid sequences. All positions containing gaps and missing data were eliminated. There were a 1097

total of 261 positions in the final dataset. Evolutionary analyses were conducted in MEGA5126

. 1098

1099

65

Discussion 1100

SGHBV produces virions containing ssDNA as efficiently as virions containing 1101

dsDNA 1102

SGHBV is the only hepadnavirus known to produce virions containing ssDNA27. 1103

We studied SGHBV alongside DHBV to gain insight into how hepadnaviruses are able 1104

to preferentially produce virions containing mature dsDNA genomes. To measure the 1105

relative ssDNA virion production we calculated the fraction of the total ssDNA molecules 1106

synthesized that were packaged in virions and divided this by the fraction of RC DNA 1107

molecules synthesized that were packaged in virions. We found this value to be 1.01 ± 1108

0.16 for SGHBV and virtually zero for DHBV. This result means that SGHBV produces 1109

virions containing ssDNA as efficiently as virions containing dsDNA. 1110

It would be interesting to know if SGHBV produces virions containing pgRNA. 1111

Because we see such high levels of ssDNA within cytoplasmic capsids and in 1112

extracellular virions, it seems unlikely that SGHBV capsids containing pgRNA are 1113

competent for virion formation and secretion. If they were competent, we would expect 1114

to see mainly RNA-containing SGHBV virions and little, to no, SGHBV virions containing 1115

DNA. This prediction assumes synthesis of ssDNA does not occur rapidly after pgRNA 1116

encapsidation, and occurs more slowly than capsid envelopment. If reverse 1117

transcription occurs rapidly, newly formed capsids containing pgRNA would very quickly 1118

become capsids containing ssDNA. Hence, it is possible that capsids containing pgRNA 1119

would not have enough time to interact with envelope proteins and be packaged in 1120

virions before reverse transcription occurs. Therefore, the best way to determine if 1121

66

SGHBV envelope proteins can productively interact with pgRNA containing capsids to 1122

form pgRNA containing virions would be to use a variant of the SGHBV P protein which 1123

lacks the ability to reverse transcribe pgRNA. In this way, capsids containing pgRNA 1124

would accumulate within the cytoplasm and it would be straightforward to determine if 1125

SGHBV can produce virions containing pgRNA. 1126

Identifying residues of Cp involved in selective production of virions containing 1127

dsDNA 1128

Using genetic complementation between SGHBV and DHBV, we found that 1129

SGHBV Cp makes a major contribution to the ability of SGHBV to produce virions 1130

containing ssDNA. This result was not surprising given the capsid maturation 1131

hypothesis. We then identified the amino acid residues of SGHBV Cp that contribute to 1132

its ability to package capsids containing ssDNA in virions. When residues 74 or 107 of 1133

DHBV Cp were changed to their SGHBV counterparts, DHBV produced virions 1134

containing ssDNA. However, changing only residue 74 of DHBV Cp led to a larger 1135

increase in ssDNA virion production, than when only residue 107 was changed. In fact, 1136

changing only residue 74 of DHBV Cp was sufficient to cause virions containing ssDNA 1137

to be secreted as efficiently as WT SGHBV Cp. In a reciprocal analysis, we changed 1138

residues 74 and 107 in SGHBV Cp to their DHBV counterparts individually and in 1139

combination. We found that changing residues 74 and 107 in combination, reduced 1140

ssDNA virion production to almost undetectable levels. In summary, we were able to 1141

change the respective behaviors of DHBV and SGHBV by making only one or two 1142

substitutions in Cp. 1143

67

These results could mean that residues 74 and 107 contribute to the appearance 1144

of the “capsid packaging signal” on the surface of capsids. It is possible that having an 1145

isoleucine at residue 74 and a glutamic acid at residue 107 causes the constitutive or 1146

early presentation of the “capsid packaging signal”, allowing capsids containing ssDNA 1147

to be enveloped and packaged in virions by DHBV envelope proteins. Determining if 1148

SGHBV capsids containing less than full-length ssDNA or pgRNA are packaged in 1149

virions would determine whether SGHBV capsids appear constitutively mature. 1150

Similarly, we could determine if capsids formed from L74I DHBV Cp or H107E DHBV 1151

Cp appear constitutively mature. If this were the case, structural or biochemical 1152

comparisons of WT DHBV capsids and the variant DHBV capsids could provide insight 1153

into the nature of the “capsid packaging signal”. 1154

Additionally, we found that when residues 74 and/or 107 of HHBV Cp were 1155

changed to their SGHBV counterparts, HHBV did not produce virions containing ssDNA. 1156

Rather, HHBV retained its ability to preferentially produce virions containing mature 1157

dsDNA. These findings suggest that additional residues, aside from 74 and 107, also 1158

dictate whether or not these hepadnaviruses selectively produce virions containing 1159

dsDNA. This result is not surprising when you consider the phylogenetic relationship 1160

between the capsid proteins from the three viruses. DHBV and SGHBV Cp are far more 1161

similar than either is to HHBV Cp (Figure 3.7). There are likely residues which are 1162

conserved in SGHBV and DHBV Cp but are different in HHBV Cp that also contribute to 1163

the ability of these proteins to produce virions containing ssDNA. These would be fairly 1164

straight forward to identify using chimeric SGHBV-HHBV Cp proteins. It would be 1165

interesting to see if changing residues 74 and 107 in Cp of an avihepadnavirus which is 1166

68

more closely related to SGHBV Cp, for example RGHBV Cp (Figure 3.7), would be 1167

sufficient to cause virions containing ssDNA to be produced. This could provide insight 1168

into other residues which contribute to the ability of these viruses to produce virions 1169

containing ssDNA or selectively produce virions containing dsDNA. 1170

Interestingly, similar analyses have been done to determine that changing 1171

residue 97 from a phenylalanine to a leucine in the human HBV capsid protein causes 1172

the production of virions containing ssDNA in cell culture100. For HBV, the amino acid 1173

change causing these different behaviors was a very conservative change and the two 1174

residues had similar hydrophobicities. This is similar to our results; when we made a 1175

highly conservative change at residue 74 in DHBV or SGHBV Cp we saw drastically 1176

different behaviors. The conservative nature of these amino-acid substitutions makes it 1177

difficult to predict how these changes are altering the ability of these hepadnaviruses to 1178

selectively produce virions containing mature dsDNA genomes. It will be interesting to 1179

determine if the mechanism by which these changes are causing the packaging of 1180

ssDNA in virions of HBV and DHBV are similar or distinct and could provide broader 1181

insights into how hepadnaviruses are able to selectively produce virions containing 1182

mature dsDNA genomes. 1183

While evidence for a direct interaction between the capsid and envelope proteins 1184

is lacking, it is possible that we have identified a surface on the exterior of the capsid 1185

that binds to envelope proteins to initiate virion morphogenesis. By changing residues 1186

74 and 107 of DHBV Cp, we may have altered the envelope interacting site on the 1187

surface of the capsid, allowing both mature and immature capsids to productively 1188

interact with envelope proteins to be packaged into a virion. 1189

69

Unfortunately there is no high resolution crystal structure of the DHBV capsid, so 1190

it is impossible to know exactly where these residues lie on the capsid. The structure of 1191

the HBV capsid has been determined47, but DHBV Cp and human HBV Cp are only 1192

weakly phylogenetically related, making it difficult to use the structure of the HBV capsid 1193

to make predictions about the DHBV capsid structure. Using a low resolution structure 1194

of DHBV capsids that has been generated using cryo-electron microscopy and 1195

subsequent 3D image reconstruction48, as well as other models of the DHBV capsid 1196

structure, made using a different approach49,127, we predict that residues 74 and 107 of 1197

DHBV Cp are at, or near, the tip of the capsid spikes. While we cannot say with 1198

certainty whether residues 74 and 107 are buried or exposed on the surface of the 1199

capsid, it is likely that residues 74 and 107 of the DHBV Cp are located on the exterior 1200

of the capsid, rather than disposed towards the lumen of the capsid. As such, these 1201

residues would be in a position to interact with the envelope proteins and/or host factors 1202

during virion morphogenesis. 1203

The role of envelope proteins in virion formation and the selective production of 1204

virions containing mature dsDNA genomes 1205

Interestingly, we found that SGHBV a determinant between residues 61 and 120 1206

of SGHBV L contributes to the production of virions containing ssDNA independent of 1207

SGHBV Cp. This was an unexpected finding because the “capsid maturation” 1208

hypothesis posits selective production of virions containing mature dsDNA genomes is a 1209

function of the capsid and, therefore, is encoded solely by Cp. These results mean that 1210

SGHBV envelope proteins have access to cytoplasmic capsids containing both mature 1211

70

and immature genomes. This observation suggests that localization of capsids to 1212

subcellular sites of envelopment is not dependent on the state of capsid maturation. 1213

A simple explanation for our finding is that (1) the residues between 61 and 120 1214

of L form a surface which interacts with cytoplasmic capsids (2) the capsid-interacting 1215

site formed by the variant SG 61-120 D L (and WT SGHBV L) is different than the 1216

capsid interacting site formed by WT DHBV envelope proteins and (3) this difference 1217

allows SG 61-120 D L (and WT SGHBV L) to facilitate the packaging of capsids into 1218

virions, independent of capsid maturation or the genome within. 1219

Another idea is that hepadnaviruses require an “envelope protein maturation” 1220

which is required for virion formation and maturation of the envelope protein would be 1221

required for it to productively bind to and envelop capsids. One example of such a 1222

change in envelope proteins could be a structural or topological change in the envelope 1223

protein. This would not be hard to imagine given the complex and multiple topologies of 1224

the L protein. For example, this change in topology could allow for membrane curvature 1225

and subsequent budding of the capsid into the secretory vesicle. 1226

Just as genome maturation triggers capsid maturation, it is possible that capsid 1227

maturation triggers envelope protein maturation. Perhaps there are two forms of the L or 1228

S envelope proteins; a “packaging incompetent” and a “packaging competent” form. 1229

Capsid maturation could cause the envelope proteins to shift to a “packaging 1230

competent” form, thereby triggering the interactions required for capsid envelopment 1231

and subsequent packaging into a virion. SGHBV envelope proteins may constitutively 1232

be in a “packaging competent” state (or location), which is why they can envelope 1233

71

capsids independent of capsid maturation and package them in virions. Further, it is 1234

possible that a determinant within amino acids 61 and 120 of SGHBV L causes L to 1235

appear constitutively “mature” or in a “packaging competent” state. 1236

We also found that SGHBV packages a larger fraction of the capsids containing 1237

RC DNA into virions when compared to DHBV. We determined that SGHBV L protein 1238

(and specifically a determinant between residues 61 and 120) is responsible for this 1239

feature. Similarly, the PreS region of DHBV L is responsible for the ability of DHBV to 1240

package a small fraction of capsids containing RC DNA into virions and its ability to 1241

preferentially produce virions containing dsDNA. 1242

One idea is that the ability to package a large fraction of the capsids containing 1243

RC DNA into virions and the ability to produce virions containing ssDNA are related and 1244

the “envelope protein maturation” we propose is a rate-limiting step in virion 1245

morphogenesis. This would be consistent with the finding that SGHBV envelope 1246

proteins contribute to its ability to package a large fraction of the RC DNA synthesized 1247

and the ability to produce virions containing ssDNA. However, we cannot rule out the 1248

possibility that the difference we observed in ability to package capsids containing RC 1249

DNA into virions may be due to differences in expression or steady-state levels of the 1250

respective envelope proteins, in addition to or rather than intrinsic differences in the 1251

properties of these proteins. The next step to understanding the L protein’s role in the 1252

selective production of virions containing mature dsDNA genomes and in dictating the 1253

proportion of capsids that get packaged into virions would be to more precisely map the 1254

determinants in L that are responsible for the respective behaviors of SGHBV and 1255

DHBV. Determining if high/low dsDNA virion production and immature/selective 1256

72

production of virions map to the same or different residue(s) could provide insight into 1257

why SGHBV and DHBV envelope proteins exhibit these different behaviors. 1258

73

1259

1260

CHAPTER 4 1261

1262

1263

IDENTIFYING AMINO ACID RESIDUES OF 1264

AVIHEPADNAVIRAL CAPSID AND ENVELOPE PROTEINS 1265

THAT CONTRIBUTE TO THE PACKAGING OF CAPSIDS INTO 1266

VIRIONS 1267

1268

Studies from this chapter will be continued and expanded upon by 1269

Dan Loeb and Karolyn Pionek 1270

74

Abstract 1271

Hepadnaviruses are enveloped dsDNA viruses whose large (L) and small (S) envelope 1272

proteins are required for virion formation. Hepadnaviruses replicate their genomes 1273

within cytoplasmic capsids, through reverse transcription. The structural subunit of the 1274

capsid is the capsid protein (Cp). It is thought that the L protein interacts with 1275

cytoplasmic capsids to (1) regulate cccDNA amplification and (2) envelope and package 1276

capsids containing dsDNA into virions. In this chapter, I describe a serendipitously- 1277

discovered inability of HHBV envelope proteins to package DHBV and SGHBV capsids 1278

into virions and illustrate how we used this as an opportunity to identify residues of Cp 1279

that are involved in capsid packaging and virion formation. We found that substituting a 1280

region between residues 69-114 of HHBV Cp into DHBV Cp was sufficient to restore the 1281

ability of HHBV envelope proteins to package DHBV capsids into virions. This suggests 1282

that a determinant between residues 69 and 114 of Cp contributes to capsid packaging 1283

into virions and possibly interacts with envelope proteins during virion morphogenesis. 1284

Within this region, DHBV Cp and HHBV Cp differ at only eleven amino acids. A similar 1285

approach can be taken to identify regions of the envelope proteins that are involved in 1286

capsid packaging/virion production. 1287

1288

75

Introduction 1289

Human hepatitis B virus is the prototype member of the Hepadnaviridae family of 1290

viruses. This family includes viruses which can infect a variety of mammals 1291

(orthohepadnaviruses) and a variety of birds (avihepadnaviruses); all have a very 1292

narrow host range19,21. All hepadnaviruses are enveloped viruses and have similar 1293

virion architectures (Figure 1.1). The virion core contains the viral polymerase protein 1294

(P) that is covalently attached to the 5’ end of the minus-strand of its dsDNA genome. 1295

The genome and P protein are enclosed within a protein shell, known as a capsid. The 1296

capsid is made up of 240 copies of the capsid protein (Cp). Around the capsid is a 1297

lipoprotein shell, known as the envelope. The envelope of the virion consists of host-1298

derived lipids and several viral envelope proteins; for avihepadnaviruses these are the 1299

large (L) and the small (S) surface proteins (Figure 1.1A). 1300

Hepadnaviruses are dsDNA viruses which replicate their genomes through 1301

reverse transcription of an RNA intermediate known as the pregenomic RNA (pgRNA). 1302

Genome replication occurs within cytoplasmic capsids. This means cytoplasmic capsids 1303

contain an array of viral replicative intermediates, ranging from pgRNA to dsDNA. A 1304

hallmark feature of hepadnaviral virion morphogenesis is that capsids containing dsDNA 1305

are preferentially packaged into virions98,121,128. Capsids containing ssDNA or pgRNA 1306

are retained within the cell. It is thought that the surface of capsids containing mature 1307

dsDNA differ from capsids containing ssDNA. This difference is thought to arise during 1308

dsDNA synthesis and is referred to as the “capsid packaging signal” or “capsid 1309

maturation signal”. This “signal” is thought to render the capsids containing dsDNA 1310

76

competent for the interactions required to be enveloped and packaged into a virion. The 1311

nature of this “signal” is not known. 1312

During virion morphogenesis, it is thought that cytoplasmic capsids containing 1313

dsDNA interact with the envelope proteins at a post-ER pre-Golgi vesicular membrane, 1314

are enveloped as they bud into this secretory vesicle and are released from the cell via 1315

a constitutive secretion pathway (for reviews, see references17,31,90). L and S are both 1316

required for virion production18,129, which suggests they play an active role in 1317

coordinating capsid envelopment/packaging into virions. 1318

Because all hepadnaviruses share a similar replication strategy, related family 1319

members have been invaluable in understanding the replication of HBV. For example, 1320

duck hepatitis B virus (DHBV) has been studied extensively to investigate many aspects 1321

of hepadnaviral biology24. In this chapter, DHBV and other avian hepadnaviruses, heron 1322

hepatitis B virus (HHBV) and snow goose hepatitis B virus (SGHBV), were used to 1323

identify regions of the capsid protein involved in virion morphogenesis. We attempted to 1324

confer the ability to produce virions containing ssDNA onto HHBV, through genetic 1325

complementation. However, instead we serendipitously discovered that HHBV envelope 1326

proteins cannot package SGHBV or DHBV capsids into virions. 1327

We saw this incompatibility between HHBV envelope proteins and SGHBV and 1328

DHBV capsids as an opportunity to identify regions of Cp and envelope proteins that 1329

contribute to virion production. When we substituted the region between amino acids 69 1330

and 114 of HHBV Cp into DHBV Cp, the ability of HHBV envelope proteins to package 1331

these capsids into virions was restored. This result indicates that a determinant between 1332

77

amino acids 69 and 114 of Cp is involved in virion production and could possibly be part 1333

of the envelope interacting site on the surface of the capsid. Within this region, DHBV 1334

Cp and HHBV Cp differ at only eleven amino acid residues; we plan to more precisely 1335

map the residue(s) involved in capsid packaging and virion production. Taking a similar 1336

strategy, we also plan to identify regions of the envelope proteins involved in virion 1337

production. 1338

78

Results 1339

HHBV envelope proteins cannot package SGHBV or DHBV capsids into virions 1340

We were able to confer the ability to efficiently package capsids containing 1341

ssDNA into virions onto DHBV by supplying SGHBV Cp to DHBV pgRNA, P, L and S 1342

(reference 130 and chapter 3 of this dissertation). We hypothesized that we could 1343

confer the ability to produce virions containing ssDNA onto HHBV using a similar 1344

strategy. To test this prediction, we co-expressed HHBV P, pgRNA and envelope 1345

proteins with SGHBV Cp in LMH cells. We did this by complementing an HHBV plasmid 1346

deficient in expressing Cp, HHBVpgRNA+P+Env+, with an SGHBV Cp donor plasmid 1347

SGHBVC+ (Figure 4.1A lanes 3 and 4). We measured the levels of RC DNA in both 1348

cytoplasmic capsids and saw no defect in cytoplasmic DNA synthesis, allowing us to 1349

evaluate virion production. 1350

It was immediately apparent that virion production was greatly reduced compared 1351

to either the HHBV or SGHBV comparisons (Figure 4.1A), but in an effort to be 1352

objective and to allow us to see intermediate levels of virion production we quantified 1353

dsDNA virion production. To quantitatively describe dsDNA virion production, we 1354

divided the amount of RC DNA packaged in virions by the sum of RC DNA found in 1355

cytoplasmic capsids and extracellular virions and multiplied this value by 100 to obtain a 1356

percentage, as shown below: 1357

% dsDNA packaged = (RC DNAvirions / (RC DNAcapsids + RC DNAvirions)) x 100 1358

For the HHBV, this value was 34 ± 11%, and for SGHBV it was 55 ± 5% (Figure 4.1A 1359

lanes 1-2 and 7-8). When we supplied SGHBV Cp to the corresponding HHBV 1360

79

components as described above, we saw that dsDNA virion production was reduced to 1361

4 ± 1% (Figure 4.1A lanes 3-4). This suggested HHBV envelope proteins poorly 1362

facilitate the packaging of SGHBV caspids into virions. 1363

To strengthen this interpretation, we measured the ability of HHBV envelope 1364

proteins to package SGHBV capsids into virions, using a different genetic 1365

complementation strategy. We co-expressed HHBV envelope proteins with SGHBV Cp, 1366

P and pgRNA in LMH cells, using plamsids HHBVEnv+ and SGHBVpgRNA+P+C+. We 1367

measured dsDNA virion production and found that it was essentially zero (Figure 4.2B 1368

lanes 2-3). This was drastically lower than the HHBV and SGHBV comparisons (Figure 1369

4.2B lane 1-2 and 5-6), where % dsDNA packaged was 30 ± 10% and 45 ± 20%, 1370

respectively. This result strengthened our interpretation that HHBV envelope proteins 1371

are not able to interact with and package SGHBV capsids into virions. Interestingly, 1372

SGHBV envelope proteins can package HHBV capsids into virions (Figure 4.1A lanes 1373

9-10, Figure 4.1B lanes 7-8) and can even confer the ability to package capsids 1374

containing ssDNA into virions onto HHBV (Figure 4.1B lanes 7-8). Hence, the 1375

incompatibility is specifically between HHBV envelope proteins and SGHBV capsids 1376

and is not reciprocal. 1377

Given the high phylogenetic similarity between SGHBV and DHBV Cp (Figure 1378

3.7) and the fact that both are distantly related to HHBV Cp, we predicted that HHBV 1379

envelope proteins would not be able to interact with and package DHBV capsids into 1380

virions either. To test this prediction, we performed experiments similar to those 1381

described above, except in place of SGHBV components, we used DHBV components 1382

(Figure 4.2 A and B). We found that HHBV envelope proteins did not package DHBV 1383

80

capsids into virions, regardless of the pgRNA or P protein present (Figure 4.2A lanes 3-1384

4, Figure 4.2B lanes 3-4). And that the incompatibility was not reciprocal; when we 1385

supplied DHBV envelope proteins to HHBV capsids we saw virions containing dsDNA 1386

were produced as efficiently as the DHBV comparisons (Figure 4.2A lanes 7-10 and 1387

4.2B lanes 5-8). This held true regardless of the origin of the pgRNA or P protein. Taken 1388

together, we conclude that HHBV envelope proteins poorly facilitate the packaging of 1389

SGHBV or DHBV capsids into virions. 1390

A determinant within amino acid residues 69 and 114 of Cp is involved in virion 1391

production 1392

One interpretation of the above results is that HHBV envelope proteins cannot 1393

interact with SGHBV or DHBV capsids because the “envelope-interacting site” on the 1394

surface of these capsids differs from the “envelope-interacting site” on HHBV capsids. If 1395

this were true, we predicted that we could substitute the portion of HHBV Cp containing 1396

the “envelope-interacting site” into SGHBV or DHBV Cp and restore the interaction 1397

between HHBV envelope proteins and these capsids. To this end, we made chimeric 1398

SGHBV-HHBV and DHBV-HHBV capsid proteins, co-expressed them with HHBV 1399

pgRNA, P, L and S and measured dsDNA virion production. 1400

We initially substituted a region between residues 22 and 139 of HHBV Cp into 1401

SGHBV Cp; within this region, SGHBV Cp and HHBV Cp differ at seventeen amino acid 1402

residues. We called this Cp variant H 22-139 SG Cp. When we co-expressed H 22-139 1403

SG Cp with HHBV P, pgRNA, L and S, we found that 31 ± 2% of the RC DNA 1404

synthesized was packaged into virions (Figure 4.1A lanes 5-6); which was not 1405

81

statistically different than the HHBV comparison, where % dsDNA production was 34 ± 1406

11% (Figure 4.1A lanes 1-2). This result indicated that substituting the region between 1407

22 and 139 into SGHBV Cp was sufficient to restore an interaction between these 1408

capsids and HHBV envelope proteins. Suggesting a determinant between amino acid 1409

residues 22 and 139 of Cp is involved in virion production and possibly interacts with the 1410

envelope proteins during capsid envelopment/packaging into virions. 1411

We more precisely mapped the region of Cp responsible for the observed 1412

species-specific incompatibility using a chimeric DHBV-HHBV Cp variant; we 1413

substituted a region between amino acids 69 and 114 of HHBV Cp into DHBV Cp and 1414

co-expressed this chimeric Cp (H 69-114 D Cp) with HHBV envelope proteins, P and 1415

pgRNA. We saw no defect in DNA synthesis, allowing us to evaluate virion production. 1416

We found that 33 ± 16% of the RC DNA molecules produced were packaged into virions 1417

(Figure 4.1A lanes 5-6); this value was not statistically different than the HHBV 1418

comparison, 34 ± 11% (Figure 4.2A lanes 1-2). Indicating a determinant within residues 1419

69 and 114 of Cp is involved in virion production; within this region, HHBV Cp and 1420

DHBV Cp differ at only eleven residues. It is possible that a determinant within this 1421

region forms a surface on the exterior of the capsid which interacts with the envelope 1422

proteins during capsid envelopment or viral egress. 1423

1424

82

1425

1426

FIGURE 4.1. HHBV envelope proteins cannot package SGHBV capsids into 1427

virions. A determinant between amino acid residues 22 and 139 of Cp contributes 1428

to virion production. 1429

Southern blot analysis. (A.) A plasmid expressing HHBV pgRNA, P, L and S was co-1430

transfected with a plasmid expressing HHBV Cp for an HHBV comparison (lanes 1 1431

83

and 2), SGHBV Cp (lanes 3 and 4) or H 22-139 SG Cp (lanes 5 and 6). A plasmid 1432

expressing SGHBV pgRNA, P, L and S was co-transfected with a plasmid expressing 1433

SGHBV Cp (lanes 7 and 8) for an SGHBV comparison or HHBV Cp (lanes 9 and 10). 1434

(B.) A plasmid expressing HHBV pgRNA, P and Cp was co-expressed with a plasmid 1435

expressing HHBV L and S (lanes 1 and 2) for an HHBV comparison or a plasmid 1436

expressing SGHBV L and S (lanes 7 and 8). A plasmid expressing SGHBV pgRNA, P 1437

and Cp was co-transfected with a plasmid expressing SGHBV L and S (lanes 5 and 6) 1438

for a SGHBV comparison or a plasmid expressing HHBV L and S (lanes 3 and 4). 1439

Below are the mean values and standard deviations for dsDNA virion production (% 1440

dsDNA pkgd); RC = relaxed circular DNA, DL = duplex linear DNA, SS = single-1441

stranded DNA, C = cytoplasmic capsid DNA, V = virion DNA. 1442

1443

84

1444

1445

FIGURE 4.2. HHBV envelope proteins cannot package DHBV capsids into virions. 1446

A determinant between amino acid residues 69 and 114 of Cp contributes to 1447

virion production. 1448

Southern blot analysis. (A.) A plasmid expressing HHBV pgRNA, P, L and S was co-transfected with a 1449

plasmid expressing HHBV Cp for an HHBV comparison (lanes 1 and 2), DHBV Cp (lanes 3 and 4) or H 1450

69-114 D Cp (lanes 5 and 6). A plasmid expressing DHBV pgRNA, P, L and S was co-transfected with 1451

85

a plasmid expressing DHBV Cp (lanes 7 and 8) for a DHBV comparison or HHBV Cp (lanes 9 and 10). 1452

(B.) A plasmid expressing HHBV pgRNA, P and Cp was co-expressed with a plasmid expressing HHBV 1453

L and S (lanes 1 and 2) for an HHBV comparison or a plasmid expressing DHBV L and S (lanes 7 and 1454

8). A plasmid expressing DHBV pgRNA, P and Cp was co-transfected with a plasmid expressing DHBV 1455

L and S (lanes 5 and 6) for a DHBV comparison or a plasmid expressing HHBV L and S (lanes 3 and 1456

4). Below are the mean values and standard deviations for dsDNA virion production (% dsDNA pkgd); 1457

RC = relaxed circular DNA, DL = duplex linear DNA, SS = single-stranded DNA, C = cytoplasmic capsid 1458

DNA, V = virion DNA. 1459

1460

86

1461

FIGURE 4.3. Region of the capsid protein (Cp) found to contribute to virion 1462

production contains the residues of Cp involved in selective production of virions 1463

containing dsDNA. 1464

Three-way alignment of DHBV3, HHBV4 and SGHBV1-15 Cp amino acid sequences. Residues found to 1465

contribute to the ability of Cp to preferentially package capsids containing dsDNA into virions are 1466

indicated by a red asterisks above the alignment. The region of Cp found to contribute to capsid 1467

packaging/virion production is indicated by a solid red line above the alignment. 1468

1469

87

Discussion 1470

In this chapter, I provide evidence that HHBV envelope proteins cannot 1471

productively interact with DHBV or SGHBV capsids to package these capsids into 1472

virions. I found that substituting the region between residues 69 and 114 of HHBV Cp 1473

into SGHBV Cp was sufficient to restore the ability of these capsids to be packaged into 1474

virions by HHBV envelope proteins. This result indicates that a determinant within 1475

amino acid residues 69 and 114 of Cp is involved in capsid packaging/virion production 1476

and possibly forms a surface on the exterior of the capsid that interacts with the 1477

envelope proteins during virion morphogenesis. Interestingly, the residues we found to 1478

be involved in the selective production of virions containing ssDNA (74 and 107) lie 1479

within this region (Figure 4.3); suggesting residues within this region of Cp form an 1480

important surface on the exterior of the capsid. 1481

Using a similar approach, regions of the envelope proteins involved in capsid 1482

packaging/virion production can be identified. For example, regions of DHBV envelope 1483

proteins can be substituted into DHBV envelope proteins and supplied to DHBV 1484

capsids, in an attempt to restore the interaction of HHBV envelope proteins with DHBV 1485

capsids and restore capsid packaging/virion production. The residues identified in L 1486

could interact with itself and/or the S protein within the membrane in which they reside, 1487

to form an interface that cytoplasmic capsids can interact with to initiate capsid 1488

envelopment and packaging into a virion. It will be interesting to see if the residue(s) of 1489

L that we find to contribute to capsid envelopment/packaging into a virion maps to the 1490

same or different residue(s) we found to be involved in the selective production of 1491

virions containing dsDNA (in Chapter 3). 1492

88

1493

1494

CHAPTER 5 1495

1496

1497

SUMMARY AND FUTURE DIRECTIONS 1498

1499

89

Chronic infection of HBV is a major health concern because it is one of the 1500

leading causes of hepatocellular carcinoma worldwide. While therapies exist, they 1501

cannot cure a chronic HBV infection. In recent years, there has been an increased 1502

interest and effort by pharmaceutical companies to come up with a cure for chronic HBV 1503

infection. To better identify antiviral drug targets and develop new treatment options that 1504

rid infected individuals of the virus, there is a need to better understand HBV replication 1505

and biology. The goal of my dissertation research was to better understand, 1506

mechanistically, how infectious viral particles are formed, with the hope that a better 1507

understanding of this process could provide insight into how to disrupt HBV replication. 1508

HBV is a double-stranded DNA (dsDNA) virus that replicates its genome through 1509

reverse transcription. Genome replication takes place entirely within cytoplasmic 1510

capsids. HBV is also an enveloped virus, which means the capsid itself is surrounded 1511

by a lipo-protein shell, known as an envelope. How the capsid acquires an envelope 1512

and is packaged into virions is incompletely understood in HBV replication. 1513

When I started graduate school, those in the field knew that capsid envelopment 1514

and virion formation do not occur in the absence of envelope proteins. This knowledge 1515

suggested that the envelope proteins actively participate in coordinating the packaging 1516

of capsids into virions by interacting with cytoplasmic capsids to facilitate their 1517

envelopment and packaging into a virion. Hepadnavirologists also knew that only 1518

capsids containing dsDNA genomes are packaged into virions, but the mechanisms 1519

underlying the ability of hepadnaviruses to selectively package capsids containing 1520

dsDNA into virions was not defined. It was thought that at some point during DNA 1521

synthesis, capsids acquire a property and/or undergo a conformational change that 1522

90

allows them to interact with envelope proteins and packaged into virions. The 1523

acquisition of this property is referred to as “capsid maturation”. 1524

Selective packaging of capsids containing mature genomes is a general 1525

characteristic of hepadnaviruses. However, one hepadnavirus, SGHBV, does not 1526

adhere to this principle. SGHBV produces virions containing ssDNA. Because of this 1527

unique feature of SGHBV, Michael Hayes, a former research technician in our lab, 1528

proposed to use SGHBV to study “capsid maturation” and selective packaging of 1529

capsids containing dsDNA into virions. He laid the foundation for much of the work I 1530

have done by obtaining a plasmid expressing SGHBV that he used to design and test 1531

several of the initial SGHBV plasmids from which all of our SGHBV plasmids were 1532

derived. 1533

The ultimate goals of my research were to better understand (1) how cytoplasmic 1534

capsids acquire an envelope and package capsids into virions and (2) how 1535

hepadnaviruses are able to discriminate between capsids containing mature dsDNA 1536

genomes and capsids containing immature ssDNA or pgRNA to selectively produce 1537

virions containing dsDNA. However, my initial goal was to determine why SGHBV lacks 1538

a mechanism for discriminating between capsids containing mature dsDNA and 1539

immature ssDNA the different types of capsids and packages both of these types of 1540

capsids into virions. As Mike Hayes proposed, if we can determine how and why 1541

SGHBV is able to produce virions containing ssDNA, we can begin to understand how 1542

all other hepadnaviruses are able to selectively produce virions containing dsDNA. 1543

91

Early in my graduate studies I determined that when SGHBV Cp and envelope 1544

proteins were combined with either DHBV or HHBV ssDNA and P, virions containing 1545

ssDNA were produced. This result meant that SGHBV Cp and/or the envelope proteins 1546

were contributing to the ability of SGHBV to produce virions containing ssDNA. My next 1547

step was to determine if any of these proteins (SGHBV Cp, L or S proteins) were 1548

sufficient to cause the production of virions containing ssDNA. To this end, I supplied 1549

SGHBV Cp or L and S to the remaining DHBV or HHBV viral components necessary to 1550

form a virion and evaluated ssDNA virion production. From these initial experiments, I 1551

uncovered several unappreciated properties of the capsid and envelope proteins of 1552

these three avian hepadnaviruses. 1553

I found that SGHBV envelope proteins and Cp independently contribute to the 1554

ability of SGHBV to produce virions containing ssDNA, as I present in Chapter 3 and in 1555

our recent publication130. I also found that HHBV envelope proteins cannot package 1556

SGHBV or DHBV capsids into virions, which I present in Chapter 4. I devised a way to 1557

use these findings to identify amino acid residues within Cp and L that are involved in 1558

(1) preferential production of virions containing dsDNA and (2) capsid packaging into 1559

virions. 1560

Identifying amino acids residues within Cp that are involved in the preferential 1561

production of virions containing dsDNA and capsid packaging into virions 1562

I found that making a highly conservative change at residue 74 in DHBV or 1563

SGHBV Cp drastically altered the behaviors of these proteins. I was able to convert 1564

DHBV Cp from a producer of virions containing dsDNA to a producer of virions 1565

92

containing ssDNA. I was similarly able to convert SGHBV Cp from a producer of virions 1566

containing ssDNA to a preferential producer of virions containing dsDNA. Indicating that 1567

residue 74 of Cp contributes to the ability of hepadnaviruses to selectively package 1568

capsids containing dsDNA, perhaps by contributing to capsid maturation or the 1569

presentation of the “capsid packaging signal” on the surface of the capsid. The fact that 1570

these amino acid changes were so conservative, suggests that the “capsid maturation 1571

signal” could be quite subtle. 1572

I found that a determinant within amino acid residues 69 and 114 of Cp is 1573

involved in virion production. I propose that this region forms a surface on the exterior of 1574

the capsid that contributes to its subsequent packaging into a virion by interacting with 1575

the envelope proteins to initiate viral morphogenesis. This finding, along with others 1576

described in Chapter 4, suggest that the L protein-capsid interactions that occur during 1577

virion morphogenesis are direct and specific. The fact that the residue(s) I determined to 1578

contribute to the selective production of virions containing ssDNA lie within residues 69 1579

and 114 of Cp, suggests that this region of Cp forms an important surface on the 1580

exterior of the capsid. Lab mates, Dan Loeb and Karolyn Pionek, were able to take over 1581

these studies and have preliminary evidence that a determinant involved in capsid 1582

packaging and virion production is at residue 69. The proximity of this residue to the 1583

residue found to be involved in selective dsDNA virion production, residue 74, suggests 1584

that these amino acid residues contribute to the formation of a very important surface on 1585

the capsid exterior. It is possible that the envelope proteins interact with these residues, 1586

and other residues nearby, during virion morphogenesis to facilitate capsid packaging 1587

and virion production. Structural determinations of DHBV capsids would provide insight 1588

93

into the location of residues 69 and 74 on the capsid. Further, comparisons of the WT 1589

DHBV capsid structure and the capsid structure formed by the DHBV Cp variant which 1590

causes capsids containing ssDNA to be packaged into virions (L74I DHBV Cp) could 1591

provide insight into the nature of the “capsid packaging signal”. 1592

Interestingly, similar analyses have been done to determine that changing 1593

residue 97 from a phenylalanine to a leucine in the human HBV capsid protein causes 1594

the production of virions containing ssDNA in cell culture100. For HBV, the amino acid 1595

change causing these different behaviors was also a very conservative change and the 1596

two residues had similar hydrophobicities. It will be interesting to determine if the 1597

mechanism by which these changes are causing the packaging of capsids containing 1598

ssDNA into virions of HBV and DHBV are similar or distinct and could provide broader 1599

insights into how hepadnaviruses are able to selectively produce virions containing 1600

mature dsDNA genomes. 1601

Identifying amino acids residues within L that are involved in the preferential 1602

production of virions containing dsDNA and capsid packaging into virions 1603

I determined that the L protein contributes to the ability of hepadnaviruses to 1604

selectively produce virions containing mature dsDNA genomes (Chapter 3 and our 1605

recent publication, reference 130). This suggests that the L protein can actively sense 1606

and select capsids containing mature dsDNA capsids to be packaged into virions, while 1607

excluding capsids containing immature ssDNA. This finding sheds new light on the 1608

mechanisms underlying virion morphogenesis and challenges the dogma that “capsid 1609

94

maturation”, and therefore Cp, is solely responsible for the selective production of 1610

virions containing mature dsDNA genomes. 1611

Thus far, the region of L that contributes to the selective production of virions 1612

containing dsDNA has been mapped to the region between residues 61 and 120. Within 1613

this region, DHBV and SGHBV differ at only seven residues. I am currently mapping 1614

which of these seven residues contribute to the ability of hepadnaviruses to selectively 1615

package capsids containing dsDNA into virions. Ultimately, these results will be 1616

submitted for publication. 1617

As I did with Cp, I wanted to determine which residues of L are involved in capsid 1618

packaging/virion formation by exploiting the species-specific incompatibility between 1619

HHBV envelope proteins and DHBV or SGHBV capsids. Unfortunately, I will not be able 1620

to complete these analyses during my time at UW. However, I initiated collaboration 1621

with two lab mates, Dan Loeb and Karolyn Pionek, who will be continuing these studies 1622

to work on this project so that it can be completed and published in a timely manner. 1623

1624

95

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