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-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

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Complete Replication of Hepatitis C Virus in Cell Culture

FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.

http://www.pnas.org/content/pnas/102/26/9294.full.pdf

Robust hepatitis C virus infection in vitro

More than 500 million people worldwide are persistently infected with the hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite many common features in the pathogenesis of HBV- and HCV-related liver disease, these viruses markedly differ in their virological properties and in their immune escape and survival strategies. This review assesses recent advances in our understanding of viral hepatitis, contrasts mechanisms of virus-host interaction in acute hepatitis B and hepatitis C, and outlines areas for future studies. NECROINFLAMMATORY A state in which there is morphological evidence of infiltration of inflammatory cells and necrosis of parenchymal

Immunology of hepatitis B virus and hepatitis C virus infection.

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.

Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.

L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.

Hepatitis C virus (HCV) envelope glycoproteins E1/E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a post-attachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.

https://www.jstor.org/stable/pdfplus/3371855.pdf

CD81 is an entry coreceptor for hepatitis C virus.

Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells We and others recently demonstrated that the C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2 In this study, we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells efficiently transinfect adjacent human liver cells Virus capture and transinfection require internalization of the SIGN–HCV pseudovirus complex In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection

https://www.pnas.org/content/pnas/101/39/14067.full.pdf

L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus

A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in subjects with prostate cancer

Robert J. Durso

Papers

L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.

CD81 is an entry coreceptor for hepatitis C virus.

L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus

A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in subjects with prostate cancer

Structural and immunogenicity studies of a cleaved, stabilized envelope trimer derived from subtype A HIV-1.