The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Abstract

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Despite these difficulties, related viruses, easier to cultivate, for instance bovine viral diarrhoea virus, have been used as surrogate models [38]. Secondly, recombinant forms of the envelope glycoproteins were used to search for viral receptors [33,39] but also as probes for antibody screening or neutralization of binding assays [40,41], to study HCV-induced receptor signalling during entry [42], to delineate E2-receptor interacting surfaces [43,44], or to draw a theoretical model for E2 structure [45]. Most frequently a soluble form of E2 produced in mammalian or insect cells and capable of inhibiting HCV entry [46] is used.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…29,30 By analogy to the related flaviviruses, it is assumed that E1 and E2 initially form heterodimers, but might rearrange into trimeric complexes required for entry. [31][32][33][34] The p7 protein, composed of two TM regions that are interconnected by a short cytoplasmic loop, 35 is a viroporin able to form hexa-and heptameric complexes serving as ion channels and required for virus assembly and release. 36,37 NS2 is a dimeric integral membrane protein, composed of two functionally and topologically distinct domains: a highly hydrophobic, N-terminal, membrane anchor domain and a C-terminal cysteine protease domain, which liberates the C-terminus of NS2 from NS3.…”

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

“…Furthermore, E1E2 is the only candidate vaccine to demonstrate broad nAb responses from vaccinated human volunteers (38,39,41,42). Soluble forms of E2 can be produced from mammalian and insect cells (20,21,59,60), and rational vaccine design has been proposed based on E2 cross-neutralizing epitopes (19). However, it is important to note that cross-neutralizing epitopes that contain E1 and E2 residues, such as those recognized by the broadly neutralizing MAbs AR4A and AR5A (15), are not represented in soluble E2 proteins.…”