CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.     

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.     

Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.     

Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.     

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.     

Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2

The tetraspanin CD81 plays an essential role in diverse cellular processes. CD81 also acts as an entry receptor for HCV through an interaction between the large extracellular loop (LEL) of CD81 and HCV glycoprotein E2. The E2-CD81 interaction also results in immunomodulatory effects in vitro. In this study, we examined the relationship between the dimeric crystal structure of the CD81 LEL and intact CD81. Using random mutagenesis, amino acids were identified that abolished dimerization of recombinant LEL in regions that were important for intermonomer contacts (F150S and V146E), salt bridge formation (K124T), and intramonomer disulfide bonding (T166I, C157S, and C190R). Two monomeric LEL mutants retained the ability to bind E2, K124T, and V146E, whereas F150S, T166I, C157S, and C190R did not. Introduction of K124T, V146E, and F150S mutations in full-length CD81 did not affect its oligomerization and the effects on E2 binding were less severe than for isolated LEL. These results suggest that the LEL has a more robust structure in the intact tetraspanin with regions outside the LEL contributing to CD81 dimerization.     

A model for the hepatitis C virus envelope glycoprotein E2

Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355-366.     

Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry.     

Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment

The envelope glycoproteins, E1 and E2, of hepatitis C virus (HCV) assemble intracellularly to form a noncovalent heterodimer that is expected to be essential for viral assembly and entry. However, due to the lack of a cell culture system supporting efficient HCV replication, it is very difficult to obtain relevant information on the functions of this glycoprotein oligomer. To get better insights into its biological and biochemical properties, HCV envelope glycoprotein heterodimer expressed by a vaccinia virus recombinant was purified by immunoaffinity. Purified E1E2 heterodimer was recognized by conformation-dependent monoclonal antibodies, showing that the proteins were properly folded. In addition, it interacted with human CD81, a putative HCV receptor, as well as with human low and very low density lipoproteins, which have been shown to be associated with infectious HCV particles isolated from patients. Purified E1E2 heterodimer was also reconstituted into liposomes. E1E2-liposomes were recognized by a conformation-dependent monoclonal antibody as well as by human CD81. Together, these data indicate that E1E2-liposomes are a valuable tool to study the molecular requirements for HCV binding to target cells.     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding

Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.     

Kinetics of HCV envelope proteins' interaction with CD81 large extracellular loop

We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2(661) HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2(661). The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.     

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.     

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.     

Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells

Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (V(H)) gene locus, leading to hypermutation in the V(H) genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the V(H) gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.     

Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.     

Early steps of the hepatitis C virus life cycle

To replicate its genome, a virus needs to cross the plasma membrane of a host cell and get access to cytosolic and/or nuclear components. For an enveloped virus, this involves binding to the plasma membrane, followed by migration of the virion to a microdomain or an endosomal vesicle where fusion between the virion envelope and a host cell membrane occurs. Increasing evidences indicate that virus entry is a tightly regulated process. Although we are still far from understanding the details of hepatitis C virus (HCV) entry, recent data show that this virus enters into target cells in a slow and complex multistep process involving the presence of several entry factors. Initial attachment of the virion may involve glycosaminoglycans and the low-density lipoprotein receptor, and it is followed by the sequential interaction with the scavenger receptor class B type I, the tetraspanin CD81 and tight junction protein Claudin-1, -6 or -9. Furthermore, the identification of EWI-2wint as a new partner of CD81 which blocks E2–CD81 interaction provides additional evidence of the complexity of the HCV entry process. The current knowledge accumulated on HCV entry is summarized in this review.     

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.     

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.     

Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor

Hepatitis C virus (HCV) or HCV-low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm(3)), intermediate-density particles (1. 12 to 1.18 g/cm(3)), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4 degrees C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of alpha-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of alpha-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.