Structural Flexibility of a Conserved Antigenic Region in Hepatitis C Virus Glycoprotein E2 Recognized by Broadly Neutralizing Antibodies

ABSTRACTNeutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses.IMPORTANCEApproximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

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Conformational Flexibility in the CD81-Binding Site of the Hepatitis C Virus Glycoprotein E2

Frontiers in Immunology ◽

10.3389/fimmu.2018.01396 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 8

Author(s):

Luisa J. Ströh ◽

Kumar Nagarathinam ◽

Thomas Krey

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Binding Site ◽

Conformational Flexibility ◽

Virus Glycoprotein ◽

Glycoprotein E2 ◽

Cd81 Binding

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Structure of Hepatitis C Virus Envelope Glycoprotein E2 Antigenic Site 412 to 423 in Complex with Antibody AP33

Journal of Virology ◽

10.1128/jvi.01939-12 ◽

2012 ◽

Vol 86 (23) ◽

pp. 13085-13088 ◽

Cited By ~ 62

Author(s):

L. Kong ◽

E. Giang ◽

T. Nieusma ◽

J. B. Robbins ◽

M. C. Deller ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Antigenic Site ◽

Virus Envelope ◽

Glycoprotein E2

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The core domain of hepatitis C virus glycoprotein E2 generates potent cross-neutralizing antibodies in guinea pigs

Hepatology ◽

10.1002/hep.28989 ◽

2017 ◽

Vol 65 (4) ◽

pp. 1117-1131 ◽

Cited By ~ 31

Author(s):

Patricia T. Vietheer ◽

Irene Boo ◽

Jun Gu ◽

Kathleen McCaffrey ◽

Stirling Edwards ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Guinea Pigs ◽

Neutralizing Antibodies ◽

Core Domain ◽

Virus Glycoprotein ◽

The Core ◽

Glycoprotein E2

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Global and local envelope protein dynamics of hepatitis C virus determine broad antibody sensitivity

Science Advances ◽

10.1126/sciadv.abb5938 ◽

2020 ◽

Vol 6 (35) ◽

pp. eabb5938 ◽

Cited By ~ 1

Author(s):

Elias H. Augestad ◽

Matteo Castelli ◽

Nicola Clementi ◽

Luisa J. Ströh ◽

Thomas Krey ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Protein ◽

Neutralizing Antibodies ◽

Scavenger Receptor ◽

Hypervariable Region ◽

Antigenic Site ◽

Conformational Space ◽

Type I ◽

Class B

Broad antibody sensitivity differences of hepatitis C virus (HCV) isolates and their ability to persist in the presence of neutralizing antibodies (NAbs) remain poorly understood. Here, we show that polymorphisms within glycoprotein E2, including hypervariable region 1 (HVR1) and antigenic site 412 (AS412), broadly affect NAb sensitivity by shifting global envelope protein conformation dynamics between theoretical “closed,” neutralization-resistant and “open,” neutralization-sensitive states. The conformational space of AS412 was skewed toward β-hairpin–like conformations in closed states, which also depended on HVR1, assigning function to these enigmatic E2 regions. Scavenger receptor class B, type I entry dependency of HCV was associated with NAb resistance and correlated perfectly with decreased virus propensity to interact with HCV co-receptor CD81, indicating that decreased NAb sensitivity resulted in a more complex entry pathway. This link between global E1/E2 states and functionally distinct AS412 conformations has important implications for targeting AS412 in rational HCV vaccine designs.

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Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2

mBio ◽

10.1128/mbio.00382-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 19

Author(s):

Ieva Vasiliauskaite ◽

Ania Owsianka ◽

Patrick England ◽

Abdul Ghafoor Khan ◽

Sarah Cole ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Binding Site ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Asymptomatic Infection ◽

Effective Vaccine ◽

Viral Glycoprotein ◽

Direct Acting Antiviral ◽

Glycoprotein E2

ABSTRACT The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like β-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a β-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design. IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development. IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development.

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Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

PLoS ONE ◽

10.1371/journal.pone.0052651 ◽

2012 ◽

Vol 7 (12) ◽

pp. e52651 ◽

Cited By ~ 15

Author(s):

George Koutsoudakis ◽

Jakub Dragun ◽

Sofia Pérez-del-Pulgar ◽

Mairene Coto-Llerena ◽

Laura Mensa ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Virus Glycoprotein ◽

Glycoprotein E2 ◽

Viral Receptors

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849 INTERPLAY BETWEEN BASIC RESIDUES OF HEPATITIS C VIRUS GLYCOPROTEIN E2 WITH LIPOPROTEINS, VIRAL RECEPTORS AND NEUTRALIZING ANTIBODIES

Journal of Hepatology ◽

10.1016/s0168-8278(12)60861-7 ◽

2012 ◽

Vol 56 ◽

pp. S331

Author(s):

G. Koutsoudakis ◽

J. Dragun ◽

S. Perez del Pulgar ◽

M. Coto-Llerena ◽

L. Mensa ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Virus Glycoprotein ◽

Glycoprotein E2 ◽

Viral Receptors

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Virus-Like Particles Containing the E2 Core Domain of Hepatitis C Virus Generate Broadly Neutralizing Antibodies in Guinea Pigs.

Journal of Virology ◽

10.1128/jvi.01675-21 ◽

2022 ◽

Author(s):

Joey McGregor ◽

Joshua M. Hardy ◽

Chan-Sien Lay ◽

Irene Boo ◽

Michael Piontek ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Guinea Pigs ◽

Neutralizing Antibodies ◽

Large Scale ◽

Broadly Neutralizing Antibodies ◽

Virus Like Particles ◽

Duck Hepatitis ◽

B Virus ◽

Glycoprotein E2

A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384-661), that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bnAbs) in comparison to the parental form (receptor-binding domain; RBD). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S and RBD-S, and Δ123A7-S and RBDA7-S in which 7 cysteines were replaced with alanines. While all four E2-S VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells, and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bnmAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (nAbs), with three of those animals generating bnAbs against 7 genotypes. Immune serum generated by animals with nAbs mapped to major neutralization epitopes located at residues 412-420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease including cirrhosis and cancer. Broadly neutralizing antibodies that recognise the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus like particles that display epitopes recognised by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

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Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05184-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1907-1915 ◽

Cited By ~ 29

Author(s):

Christoph Welsch ◽

Sabine Schweizer ◽

Tetsuro Shimakami ◽

Francisco S. Domingues ◽

Seungtaek Kim ◽

...

Keyword(s):

Molecular Dynamics ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Molecular Mechanisms ◽

Interaction Network ◽

Rna Replication ◽

Viral Fitness ◽

Dynamics Simulation ◽

Structural Flexibility ◽

Residue Interaction

ABSTRACTDrug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC50) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.

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