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

PLoS Pathogens

Bruno Robert¹  Isabelle Petitpas²  Patrick England³  Bruno Baron³  Aure Saulnier⁴  Annette Martin⁴  Laurence Damier-Piolle⁵  Félix A. Rey⁵  Carlos M. Kikuti⁵  Rajiv G. Tawar⁵  Thomas Krey⁵  Jacques d'Alayer⁶  Mats A. A. Persson⁷  Daniel X. Johansson⁷ 

[1] CEA, CNRS URA2096, Institut de Biologie et de Technologie de Saclay, Gif-sur-Yvette, France;CNRS UPR3296, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France;Institut Pasteur, CNRS URA2185, Plate-forme de Biophysique des Macromolécules et de leurs Interactions, Paris, France;Institut Pasteur, CNRS URA3015, Unité de Génétique Moléculaire des Virus à ARN, Paris, France;Institut Pasteur, CNRS URA3015, Unité de Virologie Structurale, Paris, France;Institut Pasteur, Plate-forme d'Analyse et de Microséquençage des Protéines, Paris, France;Karolinska University Hospital, Department of Medicine, Center for Molecular Medicine, Stockholm, Sweden

关键词: Membrane fusion;    Protein structure;    Protein structure prediction;    Disulfide bonds;    Crystal structure;    Polypeptides;    Protein structure comparison;    Hepatitis C virus;   

DOI  :  10.1371/journal.ppat.1000762

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY