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J. Biol. Chem., Vol. 283, Issue 39, 26340-26348, September 26, 2008

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Contribution of Redox Status to Hepatitis C Virus E2 Envelope Protein Function and Antigenicity^*

Emmanuel Fenouillet¹, Dimitri Lavillette, Silvia Loureiro^¶, George Krashias^¶, Guillemette Maurin, François-Loïc Cosset, Ian M. Jones^¶, and Rym Barbouche²

From the UMR 6184, CNRS, Université Aix-Marseille II, F-13015, France, and the Université de Lyon, F-69000, UCB-Lyon1, IFR128, INSERM, U758, Ecole Normale Supérieure de Lyon, Lyon, F-69007, France, and the ^¶School of Biological Sciences, University of Reading, Reading, RG66AJ, United Kingdom

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

Received for publication, July 9, 2008

^* This work was supported by Agence Nationale de Recherche sur le SIDA (ANRS) Grant HCV 2006–2007 (to E. F. and R. B. (sole support), and I. M. J., and F. L. C.), a grant from the Ligue Nationale Contre le Cancer (to F. L. C.), European Union Grant LSHB-CT-2004-005246 (to F. L. C.), and funds from the Wellcome Trust (to I. M. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by a fellowship from the ANRS.

¹ To whom correspondence should be addressed: Faculté de Médecine-Nord, Bd Dramard, F-13015 Marseille, France. Tel. and Fax: 33-491-69-88-47; E-mail: emmanuel.fenouillet{at}univmed.fr.

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