Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry. Hepatitis C virus (HCV)1 is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1).About 170 million individuals are infected with HCV. The predominant site of replication and probably infection is the liver, yet several studies have suggested that HCV may infect other cell types, such as B cells, monocytes/macrophages, and dendritic cells (2, 3) where it could induce immune dysfunctions. Understanding the molecular basis by which HCV targets liver cells and extra-hepatic sites is critical to unravel the mechanisms of its pathogenesis and disease chronicity. Although substantial progresses have been made on the molecular knowledge of HCV proteins and genomic replication, there is still no efficient and reliable cell culture system available to amplify this virus (4). This represents a major hurdle for the characterization of some steps of HCV lifecycle, such as viral assembly and cell entry. Several surrogate models of HCV virions have been developed to facilitate the study of the steps of cell entry. Production of HCV virus-like particles in insect cells has already been reported (5-8). Yet, although useful to analyze interactions with the cell surface, these particles were not infectious and did not permit functional investigation of the putative HCV receptors. Alternatively, generation of viral pseudotypes is one of the most widely used methods for assaying function...

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. H epatitis C virus (HCV) is a small enveloped virus containing single-stranded positive sense RNA. It infects up to 170 million people worldwide and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Although HCV accounts for only Ϸ12% of acute hepatitis in the United States, its high rate of persistence (70-80%) makes it responsible for almost half of the economic burden of this disease.A better understanding of the pathogenesis of hepatitis C and its control is hampered by certain characteristics of the virus. (i) HCV is genetically and probably serologically heterogeneous.(ii) The only animal model for HCV infection is the chimpanzee. (iii) Although the virus replicates sparingly in some cell lines, the in vitro method developed for detecting neutralizing antibodies (Nt Ab) to HCV in this system is so difficult to perform that it has not been widely used. Consequently, although the two envelope proteins of HCV, E1 and E2, have been expressed individually and as heterodimers, and Ab to them has been measured, there is no confirmation that such Ab accurately reflects the response to the virus or that it is Nt Ab.Previously, we identified Nt Ab to HCV by their ability to prevent replication of the virus in a lymphoid cell line (1, 2) or to prevent hepatitis C in chimpanzees (3, 4). These and a small number of similar studies (5) have provided, for almost a decade, the only direct evidence for Ab-mediated neutralization of HCV. Consequently, although considerable new knowledge has been gained about the cellular immune response to HCV, little is known about the role of the hum...