The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.

Chronic liver disease caused by infection with hepatitis C virus (HCV) is an important global health problem that currently affects 170 million people. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow production of viruses infectious both in vitro and in vivo . Although this improvement was important, because of the restriction to the JFH1 isolate and a single chimera consisting of J6CF and JFH1-derived sequences, broadly based comparative studies between different HCV strains were not possible. Therefore, in this study we created a series of further chimeric genomes allowing production of infectious genotype (GT) 1a, 1b, 2a, and 3a particles. With the exception of the GT3a/JFH1 chimera, efficient virus production was obtained when the genome fragments were fused via a site located right after the first transmembrane domain of NS2. The most efficient construct is a GT2a/2a chimera consisting of J6CF- and JFH1-derived sequences connected via this junction. This hybrid, designated Jc1, yielded infectious titers 100– to 1,000-fold higher than the parental isolate and all other chimeras, suggesting that determinants within the structural proteins govern kinetic and efficiency of virus assembly and release. Finally, we describe an E1-specific antiserum capable of neutralizing infectivity of all HCV chimeras.

A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175–1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfection of an HERV-W Env expression vector in a panel of cell lines derived from different species resulted in formation of syncytia in primate and pig cells upon interaction with the type D mammalian retrovirus receptor. Moreover, envelope glycoproteins encoded by HERV-W were specifically detected in placenta cells, suggesting that they may play a physiological role during pregnancy and placenta formation.

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.

In contrast to a detailed understanding of antiviral cellular immune responses, the impact of neutralizing antibodies for the resolution of acute hepatitis C is poorly defined. The analysis of neutralizing responses has been hampered by the fact that patient cohorts as well as hepatitis C virus (HCV) strains are usually heterogeneous, and that clinical data from acute-phase and long-term follow-up after infection are not readily available. Using an infectious retroviral HCV pseudoparticle model system, we studied a cohort of women accidentally exposed to the same HCV strain of known sequence. In this single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection. Neutralizing antibodies decreased or disappeared after recovery from HCV infection. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection despite the induction of cross-neutralizing antibodies in the late phase of infection. These data suggest that rapid induction of neutralizing antibodies during the early phase of infection may contribute to control of HCV infection. This finding may have important implications for understanding the pathogenesis of HCV infection and for the development of novel preventive and therapeutic antiviral strategies.