Cross-genotype characterization of genetic diversity and molecular adaptation in hepatitis C virus envelope glycoprotein genes

Investigation of the mechanisms underlying hepatitis C virus (HCV) envelope glycoprotein gene evolution will greatly assist rational development of broadly neutralizing antibody-based vaccines or vaccine components. Previously, comprehensive cross-genotype evolutionary studies of E1E2 have not been possible due to the paucity of full-length envelope gene sequences representative of all major HCV genotypes (1–6) deposited in international sequence databases. To address this shortfall, a full-length E1E2 clone panel, corresponding to genotypes of HCV that were previously under-represented, was generated. This panel, coupled with divergent isolates available via international sequence databases, was subjected to high-resolution methods for determining codon-substitution patterns, enabling a fine-scale dissection of the selective pressures operating on HCV E1E2. Whilst no evidence for positive selection was observed in E1, the E2 protein contained a number of sites under strong positive selection. A high proportion of these sites were located within the first hypervariable region (HVR1), and statistical analysis revealed that cross-genotype adaptive mutations were restricted to a subset of homologous positions within this region. Importantly, downstream of HVR1, a differential genotype-specific distribution of adaptive mutations was observed, suggesting that subtly different evolutionary pressures shape present-day genotype diversity in E2 outside HVR1. Despite these observations, it is demonstrated that purifying selection due to functional constraint is the major evolutionary force acting on HCV E1E2. These findings are important in the context of neutralizing-antibody vaccine targeting, as well as in contributing to our understanding of E1E2 function.

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Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

Journal of Virology ◽

10.1128/jvi.79.17.11095-11104.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11095-11104 ◽

Cited By ~ 201

Author(s):

Ania Owsianka ◽

Alexander W. Tarr ◽

Vicky S. Juttla ◽

Dimitri Lavillette ◽

Birke Bartosch ◽

...

Keyword(s):

Monoclonal Antibody ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Envelope Protein ◽

Hypervariable Region ◽

Neutralizing Antibody ◽

Broadly Neutralizing Antibody ◽

New Treatments ◽

Potent Neutralization

ABSTRACT Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 μg/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.

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Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

Journal of Virology ◽

10.1128/jvi.00884-09 ◽

2009 ◽

Vol 83 (22) ◽

pp. 11456-11466 ◽

Cited By ~ 22

Author(s):

Isla Humphreys ◽

Vicki Fleming ◽

Paolo Fabris ◽

Joe Parker ◽

Bodo Schulenberg ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Positive Selection ◽

Acute Infection ◽

Glycosylation Site ◽

Full Length ◽

Multiple Time ◽

Evolutionary Analysis ◽

Virus Subtype ◽

Genotype 3A

ABSTRACT Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

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Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

Protein Expression and Purification ◽

10.1016/j.pep.2014.09.009 ◽

2014 ◽

Vol 104 ◽

pp. 20-25 ◽

Cited By ~ 2

Author(s):

Sara Ortega-Atienza ◽

Laura Lombana ◽

Julián Gómez-Gutiérrez ◽

Belén Yélamos ◽

Darrell L. Peterson ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Full Length

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A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope glycoprotein and their cross-reactivity

BMC Medical Genomics ◽

10.1186/1755-8794-8-s4-s6 ◽

2015 ◽

Vol 8 (S4) ◽

Cited By ~ 2

Author(s):

Jing Sun ◽

Vladimir Brusic

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Neutralizing Antibody ◽

Cross Reactivity ◽

Systematic Analysis ◽

Broadly Neutralizing Antibody

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Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Journal of Virology ◽

10.1128/jvi.77.5.3181-3190.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3181-3190 ◽

Cited By ~ 258

Author(s):

Keril J. Blight ◽

Jane A. McKeating ◽

Joseph Marcotrigiano ◽

Charles M. Rice

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Full Length ◽

Hcv Rna ◽

Metabolic Labeling ◽

Virus Genotype ◽

Adaptive Mutations ◽

Genotype 1A

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

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Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614942113 ◽

2016 ◽

Vol 113 (45) ◽

pp. E6946-E6954 ◽

Cited By ~ 35

Author(s):

Brian G. Pierce ◽

Zhen-Yong Keck ◽

Patrick Lau ◽

Catherine Fauvelle ◽

Ragul Gowthaman ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Rational Design ◽

Neutralizing Antibody ◽

Antibody Binding ◽

Viral Envelope ◽

Viral Escape ◽

E2 Protein ◽

Antibody Recognition

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

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Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Journal of Virology ◽

10.1128/jvi.76.8.4008-4021.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4008-4021 ◽

Cited By ~ 277

Author(s):

Thomas Pietschmann ◽

Volker Lohmann ◽

Artur Kaul ◽

Nicole Krieger ◽

Gabriele Rinck ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatoma Cell ◽

Buoyant Density ◽

Full Length ◽

Structural Proteins ◽

Hepatoma Cell Line ◽

Particle Assembly ◽

Adaptive Mutations

ABSTRACT The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

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