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

Little is known about the role of Abs in determining the outcome of hepatitis C virus (HCV) infection. By using infectious retroviral pseudotypes bearing HCV glycoproteins, we measured neutralizing Ab (nAb) responses during acute and chronic HCV infection. In seven acutely infected health care workers, only two developed a nAb response that failed to associate with viral clearance. In contrast, the majority of chronically infected patients had nAbs. To determine the kinetics of strain-specific and crossreactive nAb emergence, we studied patient H, the source of the prototype genotype 1a H77 HCV strain. An early weak nAb response, specific for the autologous virus, was detected at seroconversion. However, neutralization of heterologous viruses was detected only between 33 and 111 weeks of infection. We also examined the development of nAbs in 10 chimpanzees infected with H77 clonal virus. No nAb responses were detected in three animals that cleared virus, whereas strain-specific nAbs were detected in six of the seven chronically infected animals after Ϸ50 weeks of infection. The delayed appearance of high titer crossreactive nAbs in chronically infected patients suggests that selective mechanism(s) may operate to prevent the appearance of these Abs during acute infection. The long-term persistence of these nAbs in chronically infected patients may regulate viral replication.

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Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver. Hepatitis C virus (HCV) establishes chronic infection in 3%of the world's population, resulting in a progressive liver disease that is one of the leading indications for liver transplantation. HCV has evolved several immune evasion strategies in order to persist within the infected host (15,20,40), including genetic escape from humoral immune responses (25,46). However, functional constraints may restrict antigenic change in some regions of the virally encoded E1E2 envelope glycoproteins, such as the CD81 receptor binding site (9,11,33). The observation that glycoprotein-specific antibodies from chronically infected subjects neutralize the infectivity of laboratory prototype HCV strains yet demonstrate a limited ability to control HCV replication in vivo (40) suggest that additional means of evading antibody responses may exist.How virus particles disseminate within an immune-competent host has been a relatively neglected area of study; however, it is becoming increasingly clear that viruses employ multiple strategies to infect new target cells. Diffusion through the pericellular environment or the vascular circulation introduces a rate-limiting step in virus entry and exposes particles to the humoral immune system. Consequently, a number of viruses have evolved direct cell-to-cell modes of transmission that maximize particle delivery, often in a neutralizing antibody (nAb)-resistant manner (reviewed in reference 30).We (44) and others (48) previously reported that HCV strain JFH-1 could be transmitted via cell-free and cell-to-cell routes in vitro. We extend these observations and show that disruption of HCV particle assembly or physical separation of target and producer cells ablates transmission, demonstrating that intact virions are transferred via cell-cell conta...

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

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.

In human immunodeficiency virus (HIV)-infected individuals, the proportion of circulating mononuclear cells (PBMCs) which carry HIV provirus and the number of HIV proviral sequences per infected PBMC have been matters for conjecture. Using a double polymerase chain reaction which allows the detection of single molecules of provirus and a method of quantifying the provirus molecules, we have measured provirus frequencies in infected individuals down to a level of one molecule per 10' PBMCs. As a general rule, only a small proportion of PBMCs contain provirus (median value of samples from 12 patients, one per 8,000 cells), and most if not all of the infected cells carry a single provirus molecule. The frequency of provirus-carrying cells correlated positively both with the progression of the disease and with the success with which virus could be isolated from the same patients by cocultivation methods. Of seven asymptomatic (Centers for Disease Control stage H) patients, all but one contained one provirus molecule per 6,000 to 80,000 cells; of five Centers for Disease Control stage IV patients, all but one contained one provirus molecule per 700 to 3,300 cells. When considered in conjunction with estimates of the frequency of PBMCs that express viral RNA, our results suggest that either (i) the majority of provirus-containing cells are monocytes or (ii) most provirus-containing lymphocytes are transcriptionally inactive. We also present nucleotide sequence data derived directly from provirus present in vivo which we show is not marred by the in vitro selection of potential virus variants or by errors introduced by Taq polymerase. We argue from these data that, of the provirus present in infected individuals, the proportion which is defective is not high in the regions sequenced.

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.Hepatitis C virus (HCV), the sole member of the Hepacivirus genus within the Flaviviridae, poses a global health burden, with an estimated 170 million infected individuals (according to the WHO). The majority of patients suffer a chronic infection that is associated with a progressive liver disease (1). HCV has a short positive-sense RNA genome encoding three structural (core protein, E1, and E2) glycoproteins (gps) and seven nonstructural proteins (p7 and NS2 to NS5) (40). The E1 and E2 gps interact with cell surface receptors to facilitate particle entry via low-pH and clathrin-dependent endocytosis (9,15,29,47,71). The recent discovery that the JFH-1 strain of HCV can replicate and assemble infectious particles in cultured cells (HCVcc) has allowed investigation into the viral life cycle for the first time since its identification almost 20 years ago (41,75,79).Early studies with truncated soluble HCV E2 (sE2) identified interactions with the tetraspanin CD81 and scavenger receptor class B type I (SR-BI) (56, 62). The recent availability of HCVcc and HCV pseudoparticles (HCVpp) provided the tools to validate receptor candidates. HCV entry is thought to require at least three cellular receptors: CD81, SR-BI, and the tight junction protein claudin-1 (reviewed in references 21 and 74). Other candidate components include glycosaminoglycans (5, 6, 51), low-density lipoprotein receptor (49,77), and the C...