Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes.Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.Enveloped viruses penetrate their host cells through a complex series of interactions between the viral surface and the cell membrane. This requires the attachment of the viral envelope glycoproteins to specific cell surface receptors and subsequent membrane fusion. Highly specific viral surface glycoproteins, named fusion proteins, catalyze the latter reaction by overcoming inherent energy barriers (10, 33). To date, two classes of virus fusion proteins have been defined (33): class I fusion proteins, the most well characterized of which is influenza virus hemagglutinin (HA) (62), and class II viral fusion proteins, exemplified by the E glycoprotein of tick-borne encephalitis virus (51), a flavivirus from the family Flaviviridae, and the E1 glycoprotein of Semliki Forest virus (26), an alphavirus from the Togaviridae family. Whereas their structural characteristics are markedly different, evidence suggests that class I and class II fusion proteins share an overall similar mechanism of membrane fusion (reviewed in reference 33). At an essential stage during fusion, the fusion protein bridges the gap between the viral and cell membranes by simultaneously interacting with them. The exposure and membrane insertion of a hydrophobic stretch of about 15 residues, called the "fusion peptides" or "fusion loops," mediate this crucial step (20, 54). For influenza virus HA and sev...

Hepatitis C affects about 3% of the world population, yet its current treatment options are limited to interferon-ribavirin drug regimens which achieve a 50-70% cure rate depending on the hepatitis C virus (HCV) genotype. Besides extensive screening for HCV-specific compounds, some wellestablished medicinal drugs have recently demonstrated anti-HCV effect in HCV replicon cells. One of these drugs is arbidol (ARB), a Russian-made broad spectrum antiviral agent, which we have previously shown to inhibit acute and chronic HCV infection. Here we show that ARB inhibits the cell entry of HCV pseudoparticles of genotypes 1a, 1b and 2a in a dose-dependent fashion. ARB also displayed a dose-dependent inhibition of HCV membrane fusion, as assayed by using HCV pseudoparticles (HCVpp) and fluorescent liposomes. ARB inhibition of HCVpp fusion was found more effective on genotype 1a than on genotypes 1b and 2a. In vitro biochemical studies revealed ARB association with membrane-like environments such as detergents, and with lipid membranes. This association was particularly prominent at acidic pH which is optimal for HCV-mediated fusion. Our results suggest that the affinity of ARB for lipid membranes could account for its anti-HCV actions, together with a differential level of interaction with key motifs in HCV glycoproteins of different genotypes.The hepatitis C virus (HCV) infects an estimated 3% or 170 million of the world's population, and hepatitis C is now the most frequent indication for liver transplantation. Current treatment options are limited to pegylated recombinant interferon alpha (IFN-α) in combination with ribavirin. However viremia eradication is variably achieved depending on the genotype, with only 50% of virus eradication in genotype 1-infected patients. This is clearly a problem in North America, Europe and Japan, where genotype 1 is the most prevalent genotype. HCV † This work was supported by the ANRS (Agence Nationale de Recherches sur le SIDA et les hepatites virales) to E.I.P., by the French CNRS and INSERM, by LSHB-CT-2004-005246 (COMPUVAC) to F.L.C., by the Ligue Nationale Contre le Cancer and the Rhône-Alpes Region. Y.S.B. was partially supported by the Fulbright Visiting Scholar Program. S.J.P. is supported by NIH grants RO1 DK62187 and U19 A1066328. * Corresponding author, IBCP, UMR 5086 CNRS-UCBL, 7 passage du Vercors, 69367 Lyon Cedex 07, France. Phone: ; E-mail: e.pecheur@ibcp.fr. ◊ These authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript therefore appears resistant to IFN antiviral therapy, and this is likely to be due to some factors of viral origin (1).Historically, the development of new anti-HCV drugs has been hampered due to the lack of cell culture and small animal models that are required for pre-clinical evaluations of antiviral compounds. The generation of an HCV replicon system (2) has afforded massive an...