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

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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. Little is known about the structure of the envelope glycoproteins of the hepatitis C virus (HCV), in spite of their essential role in the viral cycle of this major human pathogen. Here, we determined the connectivity of the 9 disulfide bonds formed by the strictly conserved 18 cysteines of the ectodomain of HCV glycoprotein E2. We show that this information, together with important functional data available in the literature, impose important restrictions to the possible three-dimensional fold of the molecule. Indeed, these constraints allow the unambiguous threading of the predicted secondary structure elements along the polypeptide chain onto the template provided by the crystal structures of related flavi- and alphavirus class II fusion proteins. The resulting model of the tertiary organization of E2 shows the amino acid distribution among the characteristic class II domains, places the CD81 binding site at the interface of domains I and III, and highlights the location of a candidate fusion loop.