Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells

Abstract

The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.We first reported the generation of a vesicular stomatitis virus (VSV)/hepatitis C virus (HCV) pseudotype to understand the role of the ectodomains of HCV envelope glycoproteins in relation to the initiation of virus infection (20). Since VSV particles assemble at the plasma membrane (1), we expressed chimeric HCV envelope glycoproteins (E1G and E2G) by appending the transmembrane domain and cytoplasmic tail of VSV glycoprotein at their C termini to provide membrane anchor signals. This modification allowed for the localization of the glycoproteins to the cell surface for efficient incorporation onto VSV pseudotype particles. Pseudotypes generated from either E1G or E2G which had been expressed individually displayed a low level of infectivity, while the presence of both envelope glycoproteins significantly increased the infectious titer of the pseudotype (26). Interestingly, each HCV envelope protein is suggested to have class I and/or class II fusion peptide motifs (10,11,12,31), which may contribute to the infectivity of the parent virus. Murine leukemia virus (MuLV)-and human immunodeficiency virus (HIV)-derived pseudotypes were subsequently generated from human embryonic kidney epithelial cells (293T) by the expression of an unmodified HCV envelope genomic region (5, 15). Although there are apparent differences, the overall observations from MuLV-and HIV-derived pseudotypes were similar to observations from the VSV/HCV pseudotype (8, 26). We have previously observed that the E2 glycoprotein binds to cell surface glycosaminoglycans (GAGs) (7,24). Further studies suggested that the hypervariable region 1 (HVR1) of E2 binds with heparin, and the infectivity of the VSV/HCV pseudotype is diminished in the presence of soluble GAGs or decreased after the enzymatic removal of heparinlike GAGs from the cell surface (7). A different group of investigators have suggested that HCV particles can be efficiently purified from patient sera by using heparin, and GAG binding sites are located within E2 (27, 32).The binding of E2 with a heparin-like molecule appears to facilitate VSV-deri...

Characterization of Hepatitis C Virus Interaction with Heparan Sulfate Proteoglycans

“…This is due to the unique nature of the HCV particle, which is associated with lipoproteins. Indeed, both apoE and HCV envelope glycoproteins have been proposed to play a role in HS binding (24,(26)(27)(28)(29)(30). However, after reinvestigating this question, our data support the suggestion that apoE plays a major role in HS binding, whereas the viral envelope glycoproteins present at the surface of the virion do not seem to be involved.…”

“…However, in agreement with its high level of sequence variability, HVR1 is believed to be accessible at the surface of the viral particle, which fits with the hypothesis that this region might play a role in HCV interaction with HSPGs (24,(26)(27)(28)45). We therefore reassessed the role of HVR1 in the HCV interaction with HSPGs.…”

“…apoE is involved in HCV binding to the cell surface. Due to the presence of envelope glycoproteins as well as apolipoproteins at the surface of the HCV particle, contradictory results concerning the virion determinant involved in the initial step of binding to host cells have been obtained (24,(26)(27)(28)(29)(30). Indeed, both HCV envelope glycoprotein E2 and apoE have been proposed to be the virion component involved in binding to target cells.…”

“…Following this observation, it was shown that recombinant HCV envelope glycoprotein E2 and virion-associated glycoprotein complexes interact with HSPGs, suggesting a direct contact between the viral components of the virion and HSPGs (24,25). Furthermore, E2 hypervariable region 1 (HVR1) has been proposed to contribute to this interaction (24,(26)(27)(28). However, other regions have also been proposed to be involved in interactions with HCV envelope glycoproteins (24,26).…”

Hepatitis C Virus Entry

“…This binding is thought to be mediated by E2 and to occur preferentially with highly sulfated GAGs such as heparan sulfate (HS) (51,53). Heparin (a short, very highly sulfated variant of HS produced by mast cells) and highly but not normally sulfated HS are weak inhibitors of HCVpp and HCVcc entry (37,53,54). Treatment of target cells with glycosidases also reduces HCV infectivity (37), suggesting that an interaction with GAGs does indeed promote viral entry.…”