-Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10 5 infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-a and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown (reviewed in refs 1-3). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.

HIV pseudotypes bearing native hepatitis C virus (HCV) glycoproteins (strain H and Con1) are infectious for the human hepatoma cell lines Huh-7 and PLC͞PR5. Infectivity depends on coexpression of both E1 and E2 glycoproteins, is pH-dependent, and can be neutralized by mAbs mapping to amino acids 412-447 within E2. Cell-surface expression of one or all of the candidate receptor molecules (CD81, low-density lipoprotein receptor, scavenger receptor class B type 1, and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin) failed to confer permissivity to HIV-HCV pseudotype infection. However, HIV-HCV pseudotype infectivity was inhibited by a recombinant soluble form of CD81 and a mAb specific for CD81, suggesting that CD81 may be a component of a receptor complex. Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus classified in the family Flaviviridae. Infection is often associated with chronic disease, sometimes resulting in cirrhosis and hepatocellular carcinoma. The principal site of replication is thought to be the liver, although several laboratories have suggested that HCV may infect a wider range of cell types including monocytes͞macrophages and B cells (1, 2).HCV encodes two putative envelope glycoproteins (gps), E1 and E2, which are believed to be type I integral transmembrane proteins. In vitro expression studies have shown that both gps associate to form heterodimers, which accumulate in the endoplasmic reticulum (ER), the proposed site for HCV assembly and budding (reviewed in ref.3). The lack of in vitro systems for HCV propagation has hampered biological and physiochemical studies on the virion and its mechanism(s) of cell entry, and the cellular receptors remain unknown. HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density lipoprotein receptor (LDLR) to gain entry into cells (4-6).The selective association of a virus with a target cell is usually determined by an interaction between the viral gps and specific cell-surface receptor(s) and is an essential step in the initiation of infection. Such interaction(s) often define the host range and cellular or tissue tropism of a virus and have a role in determining virus pathogenicity. In the absence of native HCV particles, truncated version(s) of the E2 gp (7, 8), E1E2 gp-liposomes (9), and virus-like particles expressed in insect cell systems (10, 11) have been used as mimics to study virus-cell interactions. Truncated soluble versions of E2 have been reported to bind specifically to human cells and were used to identify interactions with CD81 (7, 8), scavenger receptor class B type 1 (SR-B1) (12), and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) (13,14). One limitation with these studies is that they measure only HCV gp-cell attachment and not virus-mediated cell fusion. To overcome the lack of a conventional cell culture system for the propagation of infectious HCV particles, ps...

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-␣) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.An estimated 3% of the world's population is seropositive for hepatitis C virus (HCV) (32). The acute phase of infection is often subclinical; however, approximately 70% of seropositive individuals develop a chronic infection, predisposing the infected patient to the development of progressive liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma (1, 28). The current treatments for HCV infection are alpha interferon (IFN-␣) in combination with ribavirin or, more recently, a polyethylene glycol-modified form of IFN-␣; however, sustained responses are only observed in ϳ50% of treated patients, and effectiveness varies depending on the infecting HCV genotype (19).HCV has been classified within its own genus, Hepacivirus, within the family Flaviviridae, which comprises three genera of small enveloped positive-strand RNA viruses (27). The 9.6-kb genome consists of a single open reading frame (ORF) flanked by 5Ј and 3Ј nontranslated regions (NTRs) (reviewed in references 3, 4, and 16). The 5Ј NTR contains an internal ribosome entry site (IRES), mediating cap-independent translation of the ORF of ϳ3,011 amino acids. The resulting polyprotein is processed co-and posttranslationally into at least 10 individual proteins. Host signal peptidase cleavages within the N-terminal portion of the polyprotein generate the structural proteins core (C), E1, and E2. Two HCV-encoded proteases mediate downstream cleavages, liberating the nonstructural (NS) proteins involved in viral replication. The NS2-3 protease spanning the C-terminal half of NS2 and the N-terminal one-third of NS3 catalyzes autocatalytic cleavage between NS2 and NS3. The N-terminal one-third of NS3 also encodes a serine protease that functions in concert with NS4A to cleave downstream sites, while the C-terminal two-thirds harbors RNA helicase and RNA-stimulated nucleoside triphosphatase activities. The NS5B protein exhibits an RNA-dependent RNA polymerase activity. Although the N...

Hepatitis C virus (HCV) is a major cause of liver disease. Therapeutic options are limited and preventive strategies are absent. Entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen we identified epidermal growth factor receptor and ephrin receptor A2 as host co-factors for HCV entry. Blocking of kinase function by approved inhibitors broadly inhibited HCV infection of all major HCV genotypes and viral escape variants in cell culture and an animal model in vivo. Receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and membrane fusion. These results identify RTKs as novel HCV entry co-factors and uncover that kinase inhibitors have significant antiviral activity. Inhibition of RTK function may constitute a novel approach for prevention and treatment of HCV infection.

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

To define predictors of survival time in late human immunodeficiency virus type 1 (HIV-1) disease, long- and short-duration survivors were studied after their CD4+ T cells fell to

H epatitis C virus (HCV) has emerged as the major etiological agent of liver disease. Approximately 170 million individuals are infected worldwide, and the majority are at risk for developing serious progressive liver disease, with HCV being the leading indication for liver transplantation. The HCV single-stranded RNA genome encodes a single polyprotein, which is cleaved by viral and cellular proteases to produce the structural proteins; core E1 and E2 and nonstructural proteins; p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The only approved treatment for HCV infection is interferon-␣ in combination with ribavirin, which is toxic and only effective in 50% of individuals with genotype I infections. Clearly, there is a need for more effective therapies and for the development of prophylactic and/or therapeutic vaccines.Cellular and humoral responses are generated during acute infection, but they are insufficient to achieve viral clearance in the majority of individuals, with approximately 60%-80% of new infections becoming persistent. 1,2 Neutralizing antibody (nAb) responses often provide the first-line adaptive defense against infection by limiting virus spread. However, little is known about the impact of the humoral immune response on HCV pathobiology. Serum antibodies (Abs) from chronically HCVinfected individuals demonstrate broadly reactive neutralizing properties in vitro and yet fail to control viral infection in vivo. [3][4][5] The reasons for their lack of effect are poorly understood. HCV may escape neutralization by