An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry

Abstract

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ϳ2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.

Envelope-Specific IgG3 and IgG1 Responses Are Associated with Clearance of Acute Hepatitis C Virus Infection

“…Recombinant E2 (rE2) protein was derived from HCV H77 genotype 1a (GenBank accession number AF011751) and HCV UNK3a.13.6 genotype 3a (GenBank accession number AY894683). E2 regions corresponding to HCV H77 genotype 1a isolate poly-protein amino acid residues 384 to 661, introduced into pcDNA3.1 expression vector (Life Technologies, Carlsbad, CA, USA) with a C-terminal Avitag and six-histidine epitope tag (6-His) [57,58] and codon-optimised, were purchased from GeneArt (Thermo Fisher Scientific, Waltham, MA, USA).…”

Hypervariable Region 1 in Envelope Protein 2 of Hepatitis C Virus: A Linchpin in Neutralizing Antibody Evasion and Viral Entry

“…It was recently found that HVR1-mediated NAb protection could be increased even further through the binding of HVR1-specific antibodies, possibly by increased steric occlusion mediated by HVR1-bound antibody ( 137 ). The HVR1-mediated NAb protection may function in concert with other highly variable region in E2 ( 140 , 141 ), but how this interplay functions is largely unknown.…”

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

“…The majority of bNAbs target the E2 subunit, while only a few bNAbs against E1 have been isolated [ 29 , 30 , 31 , 32 ]. Soluble recombinant versions of the ectodomain of E2 maintain receptor-binding properties and are recognized by most anti-E2 bNAbs, suggesting that recombinant E2 (partly) retains its conformation [ 33 , 34 , 35 ].…”