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

• DOI: 10.1371/journal.ppat.1000762
• Publication Date: 2010-02-18T22:45:04Z
• Authors: Thomas Krey; Jacques d'Alayer; Carlos M. Kikuti; Aure Saulnier; Laurence Damier-P...; Isabelle Petitpas; Daniel X. Johansson; Rajiv G. Tawar; Bruno Baron; Bruno Robert; Patrick England; Mats A. A. Persson; Annette Martin; Félix A. Rey
• Keywords: Protein tertiary structure; CD81; Ectodomain

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