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

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apicalcanalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. at risk of developing serious progressive liver disease, with HCV being the leading indication for liver transplantation. The principal site of HCV replication is the liver; however, the cell types targeted by the virus and the frequency of infected cells are poorly defined. 1,2 The selective association of a virus with a target cell is initially defined by interactions between the viral glycoproteins and specific cell surface molecules. Recent advances have allowed us to study the mechanism of HCV entry: firstly, the development of the retrovirus pseudoparticle system [hepatitis C virus pseudoparticle (HCVpp)], which measures glycoprotein-dependent particle entry, 3,4 and secondly, the ability of the JFH-1 strain of HCV to replicate and release infectious particles in cell culture [hepatitis C virus, cell culture produced (HCVcc)]. [5][6][7] The observation that HCVpp infects human liver-derived cell lines in vitro suggests that liver-specific receptors contribute to defining HCV tropism for the liver. Recent evidence suggests that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81, 4,7-9 the scavenger receptor class B member I (SR-BI), 9-12 and the tight junction (TJ) protein Claudin 1 (CLDN1). 13 HCV glycoproteins have been reported to interact with CD81 and SR-BI. 14 Mutagenesis and antibody blocking studies suggest that the first extracellular loop of CLDN1 interacts with HCV 13 ; however, the ex-

Hepatic sinusoidal endothelial cells are unique among endothelial cells in their ability to internalize and process a diverse range of antigens. DC-SIGNR, a type 2 C-type lectin expressed on liver sinusoids, has been shown to bind with high affinity to hepatitis C virus (HCV) E2 glycoprotein. DC-SIGN is a closely related homologue reported to be expressed only on dendritic cells and a subset of macrophages and has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules. We report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show for the first time that both C-type lectins are expressed on sinusoidal endothelial cells. We confirmed that these receptors are functional by demonstrating their ability to bind HCV E2 glycoproteins. Although these lectins on primary sinusoidal cells support HCV E2 binding, they are unable to support HCV entry. These data support a model where DC-SIGN and DC-SIGNR on sinusoidal endothelium provide a mechanism for high affinity binding of circulating HCV within the liver sinusoids allowing subsequent transfer of the virus to underlying hepatocytes , in a manner analogous to DC-SIGN presentation of human immunodeficiency virus on dendritic cells.

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