Binding, entry, and immune escape mechanisms of hepatitis C virus 公开
Uebelhoer, Luke Saba (2011)
Published
Abstract
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
TABLE OF CONTENTS
ITEM PAGE
DISTRIBUTION AGREEMENT I
APPROVAL SHEET II
ABSTRACT COVER PAGE III
ABSTRACT IV
COVER PAGE V
ACKNOWLEDGMENTS VI
TABLE OF CONTENTS VII-IX
LIST OF FIGURES AND TABLES X
LIST OF ABBREVIATIONS XI
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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