Functional Mapping of Hepatitis C Virus Envelope-Heparan Sulfate Interaction

Abstract

Hepatitis C virus (HCV) is a major cause of chronic hepatitis world-wide. Although a detailed analysis of its genomic organization has led to the identification of various genetic elements, knowledge of receptor-mediated viral attachment and entry is still limited. Glycosaminoglycan (GAG) chains on cell surface proteoglycans provide initial docking sites for the binding of viruses to target cells. Recently, we have shown that cellular binding of HCV envelope glycoprotein E2 requires heparan sulfate (HS) (Barth et al. 2003, J. Biol. Chem. 278: 41003). In this study, we aimed to (i) assess the role of E1 for viral envelope-HS interaction, (ii) define the structural requirements of HS-envelope binding and (iii) further determine the functional role of HS for viral binding and entry. Using soluble GAGs and FACS analysis, we demonstrate a HS-dependent binding of E1 to HuH–7 cells. HS-dependent cellular E1 binding was confirmed by surface plasmon resonance analysis demonstrating an interaction of E1 with heparin–a homologue of highly sulfated HS (KD E1: 7.5×10–7 M). Binding of envelope glycoproteins to HuH–7 cells and human hepatocytes was strongly inhibited by fully sulfated heparin, de–2-O- and de–6-O-sulfated heparin but not de-N-sulfated heparin indicating that N-sulfation of cell surface HS plays a crucial role in HCV envelope-target cell interaction. Using HS oligosaccharides in inhibition of E1/E2 binding studies, we demonstrate that a 14 saccharide subunit represents the minimal oligosaccharide required for envelope-HS interaction. Using retroviral HCV pseudotyped particles (HCVpp) as a model for viral binding and entry, we demonstrate that HCVpp binding to HuH–7 cells is strongly inhibited by pre-incubation of HCVpp with heparin, whereas temperature-dependent HCVpp entry is only partially inhibited by heparin. In conclusion, our data indicate that highly sulfated HS represents an important cell surface molecule mediating tissue-specific attachment of the viral envelope to hepatocytes. Envelope-HS interaction is mediated by both E1 and E2 envelope glycoproteins and a specific HS configuration that includes N-sulfate and a minimum of 14 saccharide subunits.