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J. Biol. Chem., Vol. 281, Issue 7, 3909-3917, February 17, 2006

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Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes^*

Dimitri Lavillette, Birke Bartosch, Delphine Nourrisson, Géraldine Verney, François-Loïc Cosset, François Penin, and Eve-Isabelle Pécheur¹

From the IFR128 Biosciences Lyon Gerland, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Claude Bernard de Lyon, 7 Passage du Vercors, 69367 Lyon Cedex 07 and the Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.

Received for publication, September 6, 2005 , and in revised form, December 5, 2005.

^* This work was supported by the French CNRS and INSERM, by European Commission Grants LSHM-CT-2004-503359 (to F. P.), VIRGIL European Network of Excellence on Antiviral Drug Resistance and LSHB-CT-2004-005246 COMPUVAC (to F. L. C.), by the Ligue Nationale Contre le Cancer, the Rhône-Alpes Region, and by the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales. 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.

¹ To whom correspondence should be addressed: IBCP, UMR 5086 CNRS-UCBL, 7 Passage du Vercors, 69367 Lyon Cedex 07, France. Tel.: 33-4-72-72-26-44; Fax: 33-4-72-72-26-04; E-mail: e.pecheur{at}ibcp.fr.

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