Initiation of Hepatitis C Virus Infection Is Dependent on Cholesterol and Cooperativity between CD81 and Scavenger Receptor B Type I

Abstract

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.Hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses and is a major cause of chronic hepatitis and hepatocellular carcinoma (3,15). The HCV genome is a positive-strand ϳ9.6-kb RNA molecule consisting of a single open reading frame which is flanked by 5Ј and 3Ј untranslated regions (UTR). The HCV 5Ј UTR contains a highly structured internal ribosome entry site (14,50,64,78,79,86), while the 3Ј UTR is essential for replication (32,88). The HCV open reading frame encodes a single polyprotein of 3,008 to 3,037 amino acids in length that is posttranslationally processed by host and viral proteases to produce at least 10 different proteins: core protein, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (6,15,64).The host-virus interactions required during the initial steps of HCV infection are not completely understood. Prior to the development of an in vitro infectious HCV system, HCV pseudotyped retroviral particles (HCVpp), which comprise a reporter retrovirus enclosed in an envelope containing HCV glycoproteins E1 and E2, have allowed detailed analyses of the host-virus interactions required for HCVpp entry (9,10,16,30,31,34,39,46,81,83). Using a combination of HCVpp and recombinant soluble E2 glycoprotein (sE2) binding assays, a number of cellular proteins have been identified as potential candidates for HCV entry receptors, including the tetraspanin protein CD81 (61), scavenger receptor B type I (SR-BI) (68), which is a cellular protein that binds high-density lipoprotein (HDL), the low-density lipoprotein receptor (LDL-R) (1, 52), the C-type lectins L-SIGN and DC-SIGN (25,33,48,62), heparin sulfate (8), and the asialoglycoprotein receptor (67).The roles of CD81 and SR-BI as HCVpp receptors are well documented (2, 7, 9, 11, 39, 68, 91), and CD81 was recently shown to be required for cell culture-derived HCV infection (47,85,89,92). However, the extent to which SR-BI is required for HCV infection and whether it functions cooperatively with CD81 are poorly understood.Cellular cholesterol is critical for infection by many viruses (23,26,40,53,73,87). Indeed, components of the receptor complex for dengue virus, another flavivirus, are reported to be localized to cholesterol-enriched microdomains called lipid rafts, such that depletion of cholesterol by meth...

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