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

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 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...

Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles

“…These mutations reside within three discrete regions, one located in E1 and two located in E2. Interestingly, HCVpp harboring mutant E2 proteins with a single mutation of a highly conserved glycine at position 418 to alanine or aspartic acid (G418A; G418D) were heavily impaired with regard to infectivity and cell-cell fusion capacity, but they exhibited different fusion behaviors depending on whether Gly-418 had been mutated to Ala or Asp (25). We therefore introduced such mutations in the Jc1 context and analyzed glycoprotein processing and release of HCV particles, as well as their infectivity and fusion properties.…”

“…Taken together these data further confirm that the HCVcc-liposome fusion assay is dependent upon the HCV E2 glycoprotein. Single Mutation in E2 Leads to a Severe Impairment of Jc1 Infectivity and Fusion-We recently showed that specific point mutations introduced in the sequence of E1 or E2 led to severe defects in HCVpp infectivity and membrane fusion without affecting HCVpp production and incorporation of E1-E2 complexes into HCVpp (25). These mutations reside within three discrete regions, one located in E1 and two located in E2.…”

“…In addition, they are valuable reagents to characterize at the molecular level the implication of targeted regions of E1 or E2 in the fusion process. In fact, both E1 and E2 have been reported to contain fusion determinants or fusion peptide candidates (24,25), suggesting that distinct regions in both E1 and E2 may cooperate to complete the fusion process (25). However, little is known at the molecular level about the events mediating HCV membrane fusion.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Fusion between the two cell lines results in the appearance of green syncytia. Variants of this system, using on one hand the luciferase reporter gene under the control of the HIV-1 promoter and the Tat transactivator on the other hand have also been developed [118,119]. Another experimental setup relies on inducing virus fusion at the cell surface ( Figure 6B) [78,104].…”

“…Clathrin-dependent endocytosis -Internalization assay [146] -Inhibition by drugs and dominant-negative mutants -Live cell microscopy with labelled HCV [154] Low pH-mediated fusion in early endosomes -Inhibitors of endosomal acidi cation -Di erent fusion assays (see main text) Attachment and receptor interaction -Cell binding assays (CHO-SR-BI, CHO-CD81, etc) [118] -Pull-down assays [152] or ELISAs [132,153] with soluble CD81 or with heparin -Cell lines for receptor complementation (see Table 1) -Lentiviral expression vectors for the 4 speci c receptors and their homologs [104,116] H + Concanamycin A, Bafilomycin A1, Chloroquine, NH 4 Cl [69,146,147] Rab5 dominant negative mutant [145,146] Eps15 dominant negative mutant, Clathrin heavy chain siRNAs, Chlorpromazine [78,[145][146][147] Abs, Ezetimibe [32] NPC1L1 EGFR…”