Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies

Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection where transmission is potentiated by cell-cell contact. To define route(s) of transmission, HCV was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by >95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess whether cell-cell transfer of viral infectivity occurs, HCV-infected cells were cocultured with fluorescently labeled na?ve cells in the presence or absence of nAbs. Enumeration by flow cytometry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immunoglobulins from HCV(+) patients. The host cell molecule CD81 and the tight junction protein Claudin 1 (CLDN1) are critical factors defining HCV entry. Soluble CD81 and anti-CD81 abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection. CD81-negative HepG2 hepatoma cells were resistant to cell-free virus infection but became infected after coculturing with JFH-infected cells in the presence of nAb, confirming that CD81-independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-CLDN1 targets suggested that cell-cell transmission is dependent on CLDN1 expression. Conclusion: These data suggest that HCV can transmit in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route for evading nAbs.     

Hepatitis C virus receptor expression in normal and diseased liver tissue

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 apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. CONCLUSION: HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.     

CD81 is an entry coreceptor 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.     

Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus

Because of the lack of a robust cell culture system, relatively little is known about the molecular details of the cell entry mechanism for hepatitis C virus (HCV). Recently, we described infectious HCV pseudo-particles (HCVpp) that were generated by incorporating unmodified HCV E1E2 glycoproteins into the membrane of retroviral core particles. These initial studies, performed with E1E2 glycoproteins of genotype 1, noted that HCVpp closely mimic the cell entry and neutralization properties of parental HCV. Because sequence variations in E1 and E2 may account for differences in tropism, replication properties, neutralization, and response to treatment in patients infected with different genotypes, we investigated the functional properties of HCV envelope glycoproteins from different genotypes/subtypes. Our studies indicate that hepatocytes were preferential targets of infection in vitro, although HCV replication in extrahepatic sites has been reported in vivo. Receptor competition assays using antibodies against the CD81 ectodomain as well as ectopic expression of CD81 in CD81-deficient HepG2 cells indicated that CD81 is used by all the different genotypes/subtypes analyzed to enter the cells. However, by silencing RNA (siRNA) interference assays, our results show that the level of Scavenger Receptor Class-B Type-I (SR-BI) needed for efficient infection varies between genotypes and subtypes. Finally, sera from chronic HCV carriers were found to exhibit broadly reactive activities that inhibited HCVpp cell entry, but failed to neutralize all the different genotypes. In conclusion, we characterize common steps in the cell entry pathways of the major HCV genotypes that should provide clues for the development of cell entry inhibitors and vaccines.     

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture-derived HCV (HCVcc). Anti-SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI-specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81-HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV-CD81 interaction.     

Neutralizing antibody response during acute and chronic hepatitis C virus infection

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 approximately 50 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.     

A human claudin-1-derived peptide inhibits hepatitis C virus entry

Hepatitis C virus (HCV) entry is a complicated process that requires multiple host factors, such as CD81, scavenger receptor BI, claudin-1 (CLDN1), and occludin. The interaction of virus and cellular entry factors represents a promising target for novel anti-HCV drug development. In this study, we sought to identify peptide inhibitors for HCV entry by screening a library of overlapping peptides covering the four above-mentioned entry factors. An 18-amino acid peptide (designated as CL58) that was derived from the CLDN1 intracellular and first transmembrane region inhibited both de novo and established HCV infection in vitro. Unlike previously reported peptides corresponding to CLDN1 extracellular loops, CL58 did not alter the normal distribution of CLDN1 and was not cytotoxic in vitro at concentrations nearly 100-fold higher than the effective antiviral dose. The inhibitory effect of CL58 appeared to occur at a late step during viral entry, presumably after initial binding. Finally, overexpressed CL58 was able to interact with HCV envelope proteins. CONCLUSION: We identified a novel CLDN1-derived peptide that inhibits HCV entry at a postbinding step. The findings expand our knowledge of the roles that CLDN1 play in HCV entry and highlight the potential for developing a new class of inhibitors targeting the viral entry process.     

Hepatitis C virus utilizes VLDLR as a novel entry pathway

Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.Hepatitis C virus (HCV) infects more than 170 million people worldwide and is a major cause of chronic liver disease. The virus persists in 80% of infected individuals and can lead to chronic liver diseases including fibrosis, cirrhosis, steatosis, and hepatocellular carcinoma. HCV, an enveloped positive-stranded virus, enters host cells by using various host factors that function as receptors and mediate endocytosis. Several host factors, including CD81 (1), claudin-1 (CLDN1) (2), occludin (OCLN) (3), and scavenger receptor class B member I (SR-BI) (4), have been identified as receptors. Heparan sulfate glycosaminoglycan represents the first attachment site (5) before the interaction of the virus with these factors. Because all the entry factors are required for productive HCV infection, HCV entry seems to be the result of an orchestrated process involving these factors. Additionally, low-density lipoprotein receptor (LDLR) (6), Niemann-Pic C1-like 1 (NPC1L1) (7), transferrin receptor 1 (TfR1) (8), and epidermal growth factor receptor (EGFR) (9) have been shown to play a role in HCV entry. CD81 was the first factor to be identified as an HCV receptor, and it plays an important role in this process by binding with the HCV envelope glycoprotein E2 (10, 11). Indeed, CD81-deficient cell lines such as HepG2 do not permit the entry of HCV (2, 3).Recent studies have demonstrated that HCV RNA replication in Huh7.5 cells is enhanced under hypoxic conditions (12). Because the oxygen content in liver tissue in vivo is estimated to be lower (with a gradient of 9–3%) than the oxygen content under in vitro culture conditions (13), the HCV life cycle may differ significantly from that observed using in vitro culture systems. The very-low-density lipoprotein receptor (VLDLR) is induced under hypoxic conditions. In turn, this receptor enhances the uptake of low-density lipoproteins (LDLs) and very-low-density lipoproteins (VLDLs) (14), possibly through the recognition of ligands (such as apolipoprotein) that associate with the lipoproteins (15). VLDLR is a type I transmembrane lipoprotein receptor belonging to the LDLR family (16). The expression of VLDLR increased 4.2-fold and 3.5-fold in HCV cirrhotic and HCV-HCC patients, respectively, as compared with normal controls without liver disease (17). In vitro analysis has shown that during the early stage of infection HCV recognizes lipoprotein receptors such as SR-BI and LDLR on target cells via the association of the virus with apolipoprotein E (ApoE) or other related ligands (18). However, the cell lines that have been widely used for the analysis of HCV infection/replication (i.e., Huh7 and its derivatives) do not express VLDLR under conventional culture conditions (12), thereby preventing analysis of the role of VLDLR in HCV infection.The HCV particle is a lipo-viro-particle (LPV) that contains lipoprotein components such as triglycerides, apolipoprotein B-100 (ApoB), and ApoE (19, 20). Because hypoxia affects the uptake of lipoproteins and therefore might influence HCV entry and replication, we hypothesized that the HCV life cycle might be influenced by oxygen levels.Here, we elucidate the presence of a novel HCV entry pathway that uses VLDLR. Under hypoxic conditions, HCV entry into an in vitro cell-culture system was increased by up-regulating VLDLR expression. Moreover, VLDLR-mediated HCV entry was independent of the CD81-mediated HCV entry pathway.     

CD81-Receptor Associations — Impact for Hepatitis C Virus Entry and Antiviral Therapies

Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.     

Hepatitis C virus (HCV)-specific immune responses of long-term injection drug users frequently exposed to HCV

BACKGROUND: Injection drug users (IDUs) who successfully clear hepatitis C virus (HCV) have a reduced risk of developing chronic reinfection, despite their continuing exposure to the virus. To identify immunological correlates for this apparent protection, we studied HCV-specific immune responses in long-term IDUs (duration, >10 years). METHODS: HCV-specific T cell responses were assessed in proliferation, enzyme-linked immunospot (ELISPOT), interferon (IFN)-gamma secretion, and cytotoxicity assays, whereas HCV-specific antibodies were assessed in enzyme immunoassays (EIAs), chemiluminescent assays, and in vitro neutralization assays. RESULTS: HCV-specific T cell proliferation and IFN-gamma production were more common in nonviremic EIA-positive IDUs (16 [94%] of 17 IDUs) than in viremic EIA-positive IDUs (9 [45%] of 20 IDUs) (P= .003). They were also noted in 16 (62%) of 26 nonviremic EIA-negative IDUs. In contrast, 19 (90%) of 21 viremic IDUs displayed neutralizing antibodies (nAbs), compared with 9 (56%) of 16 nonviremic EIA-positive IDUs (P= .04) and 0 of 24 nonviremic EIA-negative IDUs. Nonviremic IDUs with nAbs were older (P= .0115) than those without nAbs, but these groups did not differ in terms of either injection drug use duration or HCV-specific T cell responses. CONCLUSION: The reduced risk of HCV persistence in IDUs previously recovered from HCV infection correlated with T cell responses, and prolonged antigenic stimulation appears to be required to maintain humoral responses.     

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells

Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex.     

Targeting HCV entry for development of therapeutics

Recent progress in defining the molecular mechanisms of Hepatitis C Virus (HCV) entry affords the opportunity to exploit new viral and host targets for therapeutic intervention. Entry inhibitors would limit the expansion of the infected cell reservoir, and would complement the many replication inhibitors now under development. The current model for the pathway of entry involves the initial docking of the virus onto the cell surface through interactions of virion envelope and associated low density lipoproteins (LDL) with cell surface glycosaminoglycans and lipoprotein receptors, followed by more specific utilization with other hepatocyte membrane proteins: Scavenger Receptor Class B type 1 (SR-BI), CD81, Claudin 1 (CLDN1) and Occludin (OCLN). The use of blockers of these interactions, e.g. specific antibodies, suggests that inhibition of any one step in the entry pathway can inhibit infection. Despite this knowledge base, the tools for compound screening, HCV pseudoparticles (HCVpp) and cell culture virus (HCVcc), and the ability to adapt them to industrial use are only recently available and as a result drug discovery initiatives are in their infancy. Several therapies aiming at modulating the virus envelope to prevent host cell binding are in early clinical testing. The first test case for blocking a cellular co-receptor is an SR-BI modulator. ITX 5061, an orally active small molecule, targets SR-BI and has shown potent antiviral activity against HCVpp and HCVcc. ITX 5061 has exhibited good safety in previous clinical studies, and is being evaluated in the clinic in chronic HCV patients and patients undergoing liver transplantation. Entry inhibitors promise to be valuable players in the future development of curative therapy against HCV.     

From the Cover: Identification of transferrin receptor 1 as a hepatitis C virus entry factor

Hepatitis C virus (HCV) is a liver tropic pathogen that affects ∼170 million people worldwide and causes liver pathologies including fibrosis, cirrhosis, steatosis, iron overload, and hepatocellular carcinoma. As part of a project initially directed at understanding how HCV may disrupt cellular iron homeostasis, we found that HCV alters expression of the iron uptake receptor transferrin receptor 1 (TfR1). After further investigation, we found that TfR1 mediates HCV entry. Specifically, functional studies showed that TfR1 knockdown and antibody blocking inhibit HCV cell culture (HCVcc) infection. Blocking cell surface TfR1 also inhibited HCV pseudoparticle (HCVpp) infection, demonstrating that TfR1 acts at the level of HCV glycoprotein-dependent entry. Likewise, a TfR1 small-molecule inhibitor that causes internalization of surface TfR1 resulted in a decrease in HCVcc and HCVpp infection. In kinetic studies, TfR1 antibody blocking lost its inhibitory activity after anti-CD81 blocking, suggesting that TfR1 acts during HCV entry at a postbinding step after CD81. In contrast, viral spread assays indicated that HCV cell-to-cell spread is less dependent on TfR1. Interestingly, silencing of the TfR1 trafficking protein, a TfR-1 specific adaptor protein required for TfR1 internalization, also inhibited HCVcc infection. On the basis of these results, we conclude that TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization.     

In vivo evaluation of the cross-genotype neutralizing activity of polyclonal antibodies against hepatitis C virus

Control of hepatitis C virus (HCV) infection remains a huge challenge of global medical importance. Using a variety of in vitro approaches, neutralizing antibodies (nAbs) have been identified in patients with acute and chronic hepatitis C. The exact role these nAbs play in the resolution of acute HCV infection still remains elusive. We have previously shown that purified polyclonal antibodies isolated from plasma obtained in 2003 from a chronic HCV patient (Patient H) can protect human liver chimeric mice from a subsequent challenge with the autologous HCV strain isolated from Patient H in 1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross-genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different genotypes (gt) in vivo. Following passive immunization with H06-antibodies, chimeric mice were challenged with the consensus strains H77C (gt1a), ED43 (gt4a), or HK6a (gt6a). In accordance with previous results, H06-antibodies prevented infection of chimeric mice with the autologous virus. However, the outcome of a homologous challenge is highly influenced by the amount of challenge virus injected. Depending on the viral genotype used, H06-antibodies were able to protect up to 50% of chimeric mice from a heterologous challenge. Animals in which the antibody pretreatment failed displayed a clear delay in the kinetics of viral infection. Sequence analysis of the recovered viruses did not suggest antibody-induced viral escape. CONCLUSION: Polyclonal anti-HCV antibodies isolated from a chronic HCV patient can protect against an in vivo challenge with different HCV genotypes. However, the in vivo protective efficacy of cross-genotype neutralizing antibodies was less than predicted by cell culture experiments.     

In vitro interaction between hepatitis C virus (HCV) envelope glycoprotein E2 and serum lipoproteins (LPs) results in enhanced cellular binding of both HCV E2 and LPs

Hepatitis C virus (HCV) particles in serum associate with lipoproteins (LPs), and the low-density lipoprotein receptor (LDLr) has been implicated in virus attachment and entry into cells. To clarify the basis of interactions between virus and LPs, we determined whether HCV interacts with human LPs via its envelope glycoprotein E2. The binding of serum-derived virus-like particles, HCV E2, and HCV E2-LP complexes to CD81 and LDLr was studied. Incubation of HCV E2 protein with human and bovine LPs (very low density, low density, and high density) enhanced the binding of both HCV E2 and LPs to CD4+ lymphoblastoid (MOLT-4) cells, foreskin fibroblasts, and hepatocytes. The binding of HCV E2 to MOLT-4 cells was not enhanced when it was preincubated with lipid-free apoprotein B, which suggests that E2 interacts with the lipid moiety of human lipoproteins. The LP interaction was specific for HCV E2--incubation of HIV gp120 with LPs did not enhance gp120 binding to MOLT-4 cells. The enhanced HCV E2 binding required expression of both human CD81 and LDLr. These data suggest that HCV E2 associates with LDL and that the resulting complex enhances binding of both ligands to cells, which may contribute to the finding that HCV-infected individuals have significantly lower levels of LDL than control subjects.     

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.     

A model for the study of hepatitis C virus entry

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.     

Role of low-density lipoprotein receptor in the hepatitis C virus life cycle

Hepatitis C virus (HCV) particles are known to be in complex with lipoproteins. As a result of this interaction, the low-density lipoprotein (LDL) receptor (LDLR) has been proposed as a potential entry factor for HCV; however, its implication in virus entry remains unclear. Here, we reinvestigated the role of the LDLR in the HCV life cycle by comparing virus entry to the mechanism of lipoprotein uptake. A small interfering RNA targeting the LDLR in Huh-7 cells reduced HCV infectivity, confirming that this receptor plays a role in the life cycle of HCV generated in cell culture. However, kinetics of internalization were much faster for lipoproteins than for infectious HCV particles. Furthermore, a decrease in HCV RNA replication was observed by blocking the LDLR with a specific antibody, and this was associated with an increase in the ratio of phosphatidylethanolamine to phosphatidylcholine in host cells. Nevertheless, a soluble form of the LDLR inhibited both HCV entry into the hepatocytes and its binding to the LDLR expressed on Chinese hamster ovary cells, suggesting a direct interaction between the HCV particle and the LDLR. Finally, we showed that modification of HCV particles by lipoprotein lipase (LPL) reduces HCV infectivity and increases HCV binding to LDLR. Importantly, LPL treatment also induced an increase in RNA internalization, suggesting that LDLR, at least in some conditions, leads to nonproductive internalization of HCV. Conclusion: The LDLR is not essential for infectious HCV particle entry, whereas the physiological function of this receptor is important for optimal replication of the HCV genome.     

Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders

Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the na?ve (CD27-) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that na?ve cells display a higher level of activation markers than memory (CD27+) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of na?ve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.