Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus 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 ␤-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a ␤-strand of this Ig-like domain forms an ␣-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.

Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein

“…Our computational and bioinformatics approach, in concert with various previous observations [39,51,52], provide good evidence that conformational plasticity and intrinsic disorder are defining features of the E2 glycoprotein. We sought to further verify this using biophysical analysis.…”

“…There are numerous pieces of evidence that HCV E2 exhibits high conformational plasticity [39,51,52]. For example, initial E2 crystallization attempts required the removal of HVR-1, HVR-2 and AS412, this suggests they are not conducive to ordered crystal packing and, therefore, are likely to be flexible [12,14].…”

Synergistic anti-HCV broadly neutralizing human monoclonal antibodies with independent mechanisms

“…These contrasting results may be the result of greater spatial separation between E2-bound HEPC98 and HEPC74 relative to E2-bound H77.16 and HC-11. This model is plausible, since recent studies have suggested that this region of E2 is highly flexible (39,40), so NAbs with overlapping epitopes might bind to different epitope conformations or bind with different angles of approach. The structural relationship between these adjacent neutralizing epitopes certainly warrants further investigation.…”

Designing a B Cell-Based Vaccine against a Highly Variable Hepatitis C Virus

“…Given recent evidence of mobility of key E2 epitopes associated targeted by neutralizing antibodies (Deng et al, 2014 ; Li et al, 2015 ; Meola et al, 2015 ; Kong et al, 2016 ; Vasiliauskaite et al, 2017 ), as noted above, stabilization of these epitopes is an intriguing option for future vaccine design efforts, particularly in light of promising recent studies in this regard for other viruses. Recent studies on engineering HIV SOSIP gp140 trimers include designing a stabilized closed protein conformation while decreasing exposure of non-neutralizing epitopes (de Taeye et al, 2015 ), as well as generation of new “hyperstable” SOSIP designs with engineered disulfide bonds that elicit improved neutralizing antibodies (Torrents de la Peña et al, 2017 ).…”

“…Over the past 5 years, a number of studies have helped to elucidate structural features of E1 and E2, as noted in recent reviews (Khan et al, 2015 ; Kong et al, 2015a ; Pierce et al, 2016a ). These have collectively shown that key epitopes targeted by broadly neutralizing antibodies on the “front layer” of E2, which corresponds to the CD81 binding face, exhibit structural heterogeneity, in particular E2 antigenic domain E (Kong et al, 2012 ; Li et al, 2015 ; Meola et al, 2015 ), as well as antigenic domains B and D (Kong et al, 2013 ; Deng et al, 2014 ; Keck et al, 2016b ; Vasiliauskaite et al, 2017 ). This has been underscored by a recent study, which combined experimental structural and biophysical characterization with simulations to characterize mobility of the CD81 binding region of E2 (Kong et al, 2016 ).…”