by Dimitri Lavillette — last modified 2008-01-02 20:57

Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on E1E2 viral surface glycoproteins and acidic pH. However the fusion steps, the involvement of potential fusion peptide(s) within either glycoprotein and the design of fusion inhibitors remain an area of intense investigations. Based on the similarity to other flaviviruses, it is likely that HCV harbours a class II fusion protein; however, the HCV cell entry and membrane fusion steps differ in many respects from other flaviviruses.

We have evaluated the role of six possible fusion regions identified in conserved regions within E1 and E2 HCV glycoproteins of different genotypes and subtypes. We introduced changes at amino acid predicted to be critical for the properties of fusion and analyzed the phenotypes of the mutants in assays based on infectious HCVpp. Altogether, the results gathered from cell entry, receptor binding and membrane fusion assays allowed us to distinguish between conserved domains that are likely involved in membrane fusion, one possibly being the fusion peptide (1. Lavillette et al., 2007. J Virology).

Membrane fusion was inhibited by arbidol, a broad-spectrum antiviral used in Russia as prophylaxis and treatment for influenza A and B infections. Arbidol had a direct effect on membranes, particularly at acidic pH, thereby possibly impairing the interaction of the fusion peptide with the target membrane. This correlated with a strong inhibition of arbidol on HCV cell infection and replication in a genomic full length HCV replicon cell line. By modulating membrane biology, arbidol might then have a dual action on HCV lifecycle, on cell entry and genome replication (2. Pécheur et al., 2007. Biochemistry).

The identification of the HCV fusion peptide(s) and the concerted understanding of the way to inhibit its function are clearly important to establish new antiviral strategies to combat HCV infection.

References:

1. Lavillette, D., E.-I. Pécheur, P. Donot, J. Fresquet, J. Molle, R. Corbau, M. Dreux, F. Penin, and F.-L. Cosset. 2007. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol. in press.

Abstract: 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 hepacivirus genus of the Flaviviridae family. 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 pseudo-particles (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 non-conserved 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 pseudo-particles, (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 270-284, 416-430 and 600-620) 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.

2. Pecheur, E. I., D. Lavillette, F. Alcaras, J. Molle, Y. S. Boriskin, M. Roberts, F. L. Cosset, and S. J. Polyak. 2007. Biochemical Mechanism of Hepatitis C Virus Inhibition by the Broad-Spectrum Antiviral Arbidol. Biochemistry 46:6050-6059.

Abstract: Hepatitis C affects approximately 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 well-established medicinal drugs have recently demonstrated an 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 to be more effective on genotype 1a than on genotypes 1b and 2a. In vitro biochemical studies revealed association of ARB with membranelike 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.