Binding, entry, and immune escape mechanisms of hepatitis C virus Open Access

Uebelhoer, Luke Saba (2011)

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Hepatitis C virus (HCV) infection is a global health problem, affecting more than 170
million people worldwide. Currently, no vaccine exists and treatment options are limited,
highlighting the importance of elucidating factors that lead to viral persistence. One of
the most important mechanisms of HCV pathogenesis is the high variability of its
genome. Like most small RNA viruses, HCV has an extremely high replication rate, and
the highly error prone NS5B polymerase allows for robust production of minor viral
variants that may subvert host immune responses to establish persistent infection. The
work presented here focuses on two highly variable HCV proteins, the NS3 helicase/
protease and the E2 envelope glycoprotein. First, we examine the evolution of a dominant
NS3 major histocompatibility complex (MHC) class I epitope during the acute and
chronic phases of infection in a chimpanzee through seven years of follow-up. In vitro
assessment of the fitness of viral variants that arose in vivo, as well as the host immune
response directed against these variants, indicate that genomes encoding cytotoxic T
lymphocyte (CTL) escape mutations that emerge early in infection are not necessarily
optimized for replication and are eventually replaced by variants that successfully
balance escape from cellular immune pressure and replicative fitness in the chronic phase
of infection. Second, we analyze the conserved disulfide bonding patterns in the highly
variable HCV E2 protein to determine potential impact on viral life cycle. A mutagenesis
approach identified phenotypically relevant cysteine residues of E2, and we report that
the majority of these residues are essential at early steps in viral assembly, while two
allow for low levels of egress, and one allows for high levels of viral particle formation
and secretion but ablates CD81 coreceptor binding. In parallel to these experiments, a
system using high-resolution deconvolution microscopy was developed to aid in future
binding and entry studies of relevant HCV mutants. We additionally report a recombinant
chimeric antibody technique for the delivery of epitopes to antigen-specific T cells. This
work highlights the competing forces of viral infection and the immune system, and
presents a novel attenuated HCV vaccine candidate.

Table of Contents












CHAPTER 1: Introduction 1

Hepatitis C virus: a deadly human pathogen

Epidemiology, impact, significance 1

The viral life cycle 2

Binding and entry3

Fusion and uncoating 9

RNA replication, genome translation, processing 10

Virion maturation and egress 23

Current and future therapies 25

Model systems to study hepatitis C virus

Initial approaches: biochemistry and chimpanzees 29

Subgenomic and full-length replicons 30

Glycoprotein expression systems, vesicular stomatitis

virus-pseudotyping, virus-like particles 31

Hepatitis C virus pseudoparticles 32

Hepatitis C virus full-length infectious cell culture systems 33

The future of hepatitis C virus model systems 34

Hepatitis C virus and the host immune response

Onset of disease, potential outcomes, severity of infection 37

Incubation phase of infection 38

Innate immune response and viral evasion 38

Hepatocytes 38

Dendritic cells 39

Natural killer cells 41

Acute phase of infection 41

Humoral immune response 42

T cell-mediated immune response 43

Chronic phase of infection 47

Persistent antigen stimulation, anergy, deletion of T cells 48

IL-10-mediated suppression of T cell responses, induction

of regulatory T cells 49

Mutational escape of CD4+ and CD8+ T cell epitopes 51

Lymphocytic choriomeningitis virus: a surrogate model of chronic

viral infections

Pathogenesis in humans and rodents 55

Genome organization 56

Acute versus chronic variants 57

Host immune response 58

Humoral and cellular immune response 58

Memory T cell response 60

B cells as antigen-presenting cells

Antigen-presenting cell transfer as therapy 62

CD40-activation in B cell priming of T cells 63

Naïve B cell-T cell interactions 64

Recombinant chimeric antibodies: a novel system

to probe APC-T cell interactions 64

References 67

CHAPTER 2: Stable cytotoxic T cell escape mutation in hepatitis C virus

is linked to maintenance of viral fitness 130

Abstract 131

Introduction 133

Materials and methods 136

Results 146

Discussion 157

Acknowledgments 163

Figures 164

Figure legends 171

References 176

CHAPTER 3: Disruption of a conserved disulfide bond in HCV E2 protein

abrogates infectivity and impacts CD81 binding 184

Abstract 185

Introduction 186

Materials and methods 190

Results 199

Discussion 209

Acknowledgments 215

Figures 216

Figure legends 221

References 224

CHAPTER 4: Binding and entry dynamics of hepatitis C virus and

recombinant ectodomain E2 glycoprotein in human hepatoma cell lines 229

Abstract 230

Introduction 231

Materials and Methods 234

Preliminary results 240

Discussion and Future Directions 244

Figures 248

Figure Legends 256

References 262

CHAPTER 5: A novel recombinant chimeric antibody system to deliver

CD4+ T cell epitopes to naïve B cells during chronic infection 268

Abstract 269

Introduction 270

Materials and methods 274

Results 282

Discussion 291

Acknowledgments 296

Figures 297

Figure legends 302

References 305

CHAPTER 6: Conclusions and future directions 312

Figures 329

Figure Legends 331

References 332

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