Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.Hepatitis C virus (HCV) infects about 170 million people around the world. Despite the importance of HCV as a human pathogen, little is known about its cell biology. The virus was identified and cloned more than 15 years ago (7), but the lack of a robust system allowing for the production of HCV in cell culture has hampered for many years functional studies on HCV infection.In recent years, two major advances have made it possible to investigate HCV entry. A first advance has been the production of infectious retroviral particles pseudotyped with HCV envelope glycoproteins (3,14,24). Using this system of HCV pseudoparticles (HCVpp), observations on receptor usage and the facilitating role of high-density lipoprotein during entry were reported (3, 24, 55). A second major advance has been the recent development of a cell culture model for HCV (30,56,59). This system allows for the production of virus that can be efficiently propagated in cell culture (HCVcc). Therefore, the cell entry of HCV can now be investigated in the context of an infectious cycle.HCV belongs to the Hepacivirus genus in the Flaviviridae family, which also includes the Flavivirus and Pestivirus genera (31). The HCV genome encodes three structural proteins, capsid protein C and envelope glycoproteins E1 and E2, which are associated in the form of a heterodimer (13). Several cellular proteins were reported to interact in vitro with isolated E2. These putative receptors include the tetraspanin CD81 (42), the scavenger receptor class B type I (SR-BI) (50), the lectins L-SIGN and DC-SIGN (18, 32), the asialoglycoprotein receptor (49), and heparan-sulfate proteoglycans (2). The low-density lipoprotein receptor was also proposed as a candidate receptor (1). The importance of CD81 and SR-BI in HCV entry was confirmed with HCVpp (3, 4, 24) as well as with HCVcc for CD81 (30,56). Beyond receptor bind...
Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.Hepatitis C virus (HCV), a recently identified agent of parenterally transmitted non-A non-B hepatitis, causes the vast majority of transfusion-associated cases of hepatitis and a significant proportion of community-acquired hepatitis (for review, see refs. 1 and 2). HCV infection results in varied clinical outcomes, and chronic infections are common and have been associated with an increased incidence of hepatocellular carcinoma. Although a interferon has been partially successful in inhibiting HCV replication, reliable therapeutic agents for controlling or eradicating HCV infection are currently lacking.
Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, El (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of El is associated with E2 and E2-NS2 via disulfide linkages.
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