In silico directed mutagenesis identifies the CD81/claudin‐1 hepatitis C virus receptor interface

Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin‐1 and occludin. We previously reported a role for CD81/claudin‐1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin‐1 using a combination of bioinformatic sequence‐based modelling, site‐directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin‐1 to have a flexible beta conformation and identifies a motif between amino acids 62–66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin‐1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCVglycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.     

Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.     

Role of lipid metabolism in hepatitis C virus assembly and entry

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very‐low‐density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR‐BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.     

Hepatitis C virus is primed by CD81 protein for low pH-dependent fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.     

Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.     

Hepatitis C virus induces CD81 and claudin-1 endocytosis

Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.     

Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR.     

StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.     

Different domains of CD81 mediate distinct stages of hepatitis C virus pseudoparticle entry

The CD81 tetraspanin was first identified as a hepatitis C virus (HCV) receptor by its ability to bind the soluble ectodomain of envelope glycoprotein E2 (sE2). More recently, it has been suggested that CD81 is necessary but not sufficient for HCV entry into target cells. Here we present further evidence that putative human hepatocyte-specific factors act in concert with CD81 to mediate sE2 binding and HCV pseudoparticle (HCVpp) entry. Moreover, we show that CD81-mediated HCVpp entry entails E2 binding to residues in the large extracellular loop as well as molecular events mediated by the transmembrane and intracellular domains of CD81. The concept that CD81 receptor function progresses in stages is further supported by our finding that anti-CD81 monoclonal antibodies inhibit HCVpp entry by different mechanisms. The half-life of CD81-HCVpp binding was determined to be approximately 17 min, and we propose that binding is followed by CD81 oligomerization, partitioning into cholesterol-rich membrane domains, or other, lateral protein-protein interactions. This results in the formation of a receptor-virus complex that undergoes endocytosis and pH-dependent membrane fusion.     

Mobile genetic elements in the genome of the beneficial rhizobacterium Pseudomonas fluorescens Pf-5

Background

Three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for Plasmodium infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM).

Results

In our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels.

Conclusion

In contrast to Plasmodium infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.     

Mathematical model of viral kinetics in vitro estimates the number of E2-CD81 complexes necessary for hepatitis C virus entry

Interaction between the hepatitis C virus (HCV) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells. The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally. Using the recently developed cell culture systems that allow persistent HCV infection in vitro, the dependence of HCV entry and kinetics on CD81 expression has been measured. We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry. Here, we constructed a mathematical model of HCV viral kinetics in vitro, in which we accounted explicitly for the dependence of HCV entry on CD81 expression. Model predictions of viral kinetics are in quantitative agreement with experimental observations. Specifically, our model predicts triphasic viral kinetics in vitro, where the first phase is characterized by cell proliferation, the second by the infection of susceptible cells and the third by the growth of cells refractory to infection. By fitting model predictions to the above data, we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells. We found that depending on the E2-CD81 binding affinity, between 1 and 13 E2-CD81 complexes are necessary for HCV entry. With this estimate, our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels, indicating that the estimate is robust. Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction. Further, our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection.     

Viral cell recognition and entry.

Rhinovirus infection is initiated by the recognition of a specific cell-surface receptor. The major group of rhinovirus serotypes attach to intercellular adhesion molecule-1 (ICAM-1). The attachment process initiates a series of conformational changes resulting in the loss of genomic RNA from the virion. X-ray crystallography and sequence comparisons suggested that a deep crevice or canyon is the site on the virus recognized by the cellular receptor molecule. This has now been verified by electron microscopy of human rhinovirus 14 (HRV14) and HRV16 complexed with a soluble component of ICAM-1. A hydrophobic pocket underneath the canyon is the site of binding of various hydrophobic drug compounds that can inhibit attachment and uncoating. This pocket is also associated with an unidentified, possibly cellular in origin, "pocket factor." The pocket factor binding site overlaps the binding site of the receptor. It is suggested that competition between the pocket factor and receptor regulates the conformational changes required for the initiation of the entry of the genomic RNA into the cell.     

丙型肝炎病毒E2蛋白对HepG2细胞MAPK／ERK的激活

人CD81是丙型肝炎病毒（hepatitis Cvirus,HCV）的细胞表面特异性受体,HCV包膜蛋白－2（E2）可与其结合。细胞个信号调节激酶（extracellular signal-regulated protein kinase,MAPK/ERK1,2）信号途径主要介导细胞增殖及分化。为探讨HCV E2蛋白与人CD81结合对MAPK／ERK活性变化的影响,以HCV E2蛋白刺激HepG2细胞,采用免疫印迹、免疫组化及免疫荧光等方法动态观察细胞内MAPK／ERK的激活情况,并以流式细胞术检测细胞表面CD81的表达。结果表明：HepG2细胞高表达人CD81;HCV E2蛋白可激活细胞内MAPK／ERK;MAPK／ERK的磷酸化反应与HCV E2蛋白浓度、作用时间呈依赖关系;HCV E2－CD81相互作用引发的细胞异常信号转导可能与HCV致病性相关。     

EWI‐2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry

CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin‐enriched areas in the plasma membrane. In our study, we combined single‐molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI‐2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI‐2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI‐2wint promotes CD81 clustering and confinement in CD81‐enriched areas. In addition, we showed that EWI‐2wint influences the colocalization of CD81 with Claudin‐1 – a co‐receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI‐2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.     

Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.     

Hepatoma polarization limits CD81 and hepatitis C virus dynamics

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real‐time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C‐terminus, we studied the dynamics of a mutant CD81 lacking a C‐terminal tail (CD81_ΔC) and its effect(s) on HCVpp mobility and infection. CD81_ΔC showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild‐type protein in non‐polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81_ΔC expressing non‐polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization‐dependent manner.     

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry

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.     

Dynamics of cholesterol exchange in the oxysterol binding protein family

The oxysterol-binding protein-related protein (ORP) family is essential to sterol transfer and sterol-dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol-bound states. In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

CD81 is required for hepatitis C virus glycoprotein-mediated viral infection

CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.