Hepatitis C Virus E2 Protein Research Articles

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Functional selection of hepatitis C virus envelope E2-binding Peptide ligands by using ribosome display.

Small peptides that inhibit the hepatitis C virus (HCV) at the stage of viral entry have the potential to serve as attractive antiviral drugs. Ribosome display is a cell-free system for in vitro selection of peptides from large random peptide libraries. Thus, we utilized a ribosome display library technique for affinity selection of HCV envelope protein E2-binding peptide ligands. Through 13 rounds of selection, the ribosome display system generated high-affinity 12-mer peptides, and the selected peptide PE2D (MARHRNWPLVMV) demonstrated the highest specificity and affinity to the HCV E2 protein. Furthermore, amino acids 489 to 508 (YPPRPCGIVPAKSVCGPVYC) of E2 were identified as crucial for binding to PE2D. The selected peptides, especially PE2D, not only dramatically blocked E2 protein binding to hepatocytes but also dramatically inhibited HCV cell culture (HCVcc) entry into hepatocytes. HCVcc and HCV particles from HCV patient serum samples could also be specifically captured using PE2D. Our study demonstrates that the newly selected peptide ligand PE2D holds great promise for developing a new molecular probe, a therapeutic drug specifically for HCV, or an early-diagnostic reagent for HCV surface envelope antigen E2.

Conservation of Hydrophobicity within Viral Envelope Glycoproteins Reveals a Putative Hepatitis C Virus Fusion Peptide

The mechanism(s) by which hepatitis C virus (HCV) enters and infects cells remains unknown. Identifying the HCV fusion peptide(s) and understanding the early stages of infection may provide new opportunities for improved antiviral therapy. The HCV envelope glycoprotein E2 is thought to be a class II fusion protein. Class II fusion proteins are exemplified by the E protein of the tick-borne encephalitis virus (TBEV) and the E1 protein of the Semliki Forest virus (SFV). Analysis of the hydrophobicity profiles of four HCV E2 envelope glycoproteins revealed a region with a conserved three-pronged pattern of hydrophobicity, termed the tridentate (TD) region. The primary sequence of the TD region is highly conserved in all 490 HCV strains currently reported. The known fusion peptide loops of TBEV and SFV share the characteristic TD region hydrophobicity profile and significant sequence conservation in the TD region was identified in the E and E1 glycoproteins of members of the Flaviviridae and Togaviridae families, respectively. The HCV TD region peptides have membranotropic activity; in molecular dynamics (MD) simulations, the HCV TD region peptides insert into in a biomimetic bilayer in a similar manner to the TBEV fusion peptide and the peptides induce effective mixing of lipid membranes in a liposome fusion assay. Together these results indicate that the highly conserved TD region of the HCV E2 protein is a fusion peptide candidate and may be an important factor in the class II fusion mechanism.

The heat shock cognate protein 70 is associated with hepatitis C virus particles and modulates virus infectivity†

There is growing evidence that virus particles contain host cell proteins. These proteins may provide viruses with means to evade the immune system or with mechanisms for cell entry and release. A proteomic analysis performed on highly purified hepatitis C virus (HCV) J6/JFH virions identified the heat shock cognate protein 70 (HSC70) as part of the viral particles. These results were further validated via immunogold electron microscopy. The HSC70 interaction HPD motif was found present on the E2 envelope of the J6/JFH strain, as well as in over 50% of genotype 2 clinical HCV isolates. In addition, HSC70 was found associated with viral particles from an HCV genotype 2a-infected patient. Preincubation of HCV particles with anti-HSC70 antibodies decreased viral infectivity. Within infected cells, colocalization of HSC70 with the HCV core and E2 proteins was observed around lipid droplets. Reduction of HSC70 expression using an RNA interference approach decreased the volume of lipid droplets as well as viral release without affecting HCV replication levels. These results suggest that HSC70 modulates HCV infectivity and lipid droplet-dependent virus release.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

Analysis of a conserved RGE/RGD motif in HCV E2 in mediating entry

BackgroundHepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors. It is clear that HCV uses a multi-receptor complex to gain entry into susceptible cells, however key elements of this complex remain elusive. In this study, the role of a highly conserved RGE/RGD motif of HCV E2 glycoprotein in viral entry was examined. The effect of each substitution mutation in this motif was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E2 proteins, CD81 binding profiles, and conformation of mutants were examined.ResultsBased on these characteristics, mutants either displayed wt characteristics (high infectivity [≥ 90% of wt HCVpp], CD81 binding, E1E2 expression, and incorporation into viral particles and proper conformation) or very low infectivity (≤ 20% of wt HCVpp). Only amino acid substitutions of the 3rd position (D or E) resulted in wt characteristics as long as the negative charge was maintained or a neutral alanine was introduced. A change in charge to a positive lysine, disrupted HCVpp infectivity at this position.ConclusionAlthough most amino acid substitutions within this conserved motif displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, and incorporation into HCVpp. Our results suggest that although RGE/D is a well-defined integrin binding motif, in this case the role of these three hyperconserved amino acids does not appear to be integrin binding. As the extent of conservation of this region extends well beyond these three amino acids, we speculate that this region may play an important role in the structure of HCV E2 or in mediating the interaction with other factor(s) during viral entry.

Antigenic structure of the hepatitis C virus envelope 2 protein

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.

A molecular chaperone glucose-regulated protein 94 blocks apoptosis induced by virus infection

The hepatitis C virus (HCV) E2 protein has been shown to block apoptosis and has been suggested to facilitate persistent infection of the virus. Here, we report that the anti-apoptotic activity of E2 is mediated by activation of nuclear factor kappa B (NF-kappaB) that directs expression of survival gene products such as tumor necrosis factor (TNF-alpha) receptor-associated factor 2 (TRAF2), X-chromosome-linked inhibitor of apoptosis protein (XIAP), FLICE-like inhibitory protein (FLIP), and survivin. Increased levels of these proteins were observed in HCV-infected cells and a cell line producing HCV E2 protein. The activation of NF-kappaB was mediated by HCV-E2-induced expression of the molecular chaperone glucose-regulated protein 94 (GRP94). Overexpression of GRP94 alone resulted in expression of anti-apoptotic proteins and blocked apoptosis induced by tumor-necrosis-related apoptosis-inducing ligand (TRAIL). Interestingly, increased levels of GRP94 were observed in cells supporting HCV proliferation that originated from liver tissues from HCV patients. Moreover, small interfering RNA (siRNA) knock-down of GRP94 nullified the anti-apoptotic activity of HCV E2. These data indicate that HCV E2 blocks apoptosis induced by HCV infection and the host immune system through overproduction of GRP94, and that HCV E2 plays an important role in persistent HCV infection.

Replication-Competent Recombinant Vesicular Stomatitis Virus Encoding Hepatitis C Virus Envelope Proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.

Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma

Neutralizing antibodies directed against hepatitis C virus (HCV) are present in Igs made from anti-HCV-positive plasma. However, these HCV-specific Igs are largely ineffective in vivo. The mechanism for the poor effectiveness is currently unknown. We hypothesize that the presence of nonneutralizing antibodies in HCV-specific Igs interferes with the function of neutralizing antibodies, resulting in the reduction or blockage of their effect. In the present study, we identified at least two epitopes at amino acid residues 412–419 (epitope I) and 434–446 (epitope II), located downstream of the hypervariable region I within the HCV E2 protein. We demonstrated that epitope I, but not epitope II, was implicated in HCV neutralization and that binding of a nonneutralizing antibody to epitope II completely disrupted virus neutralization mediated by antibody binding at epitope I. The dynamic interaction between nonneutralizing and neutralizing antibodies may thus play a key role in determining the outcomes of HCV infection. Further exploration of this interplay should lead to a better understanding of the mechanisms of neutralization and immune escape and may indicate pathways for the manufacture of an effective HCV-specific Ig product for immune prophylaxis of HCV infection.

Multimeric HCV E2 protein obtained from pichia pastoris cells induces a strong immune response in mice

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2680) was produced in Pichia pastoris cells. E2680, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulfide-linked multimers. Both monomeric and oligomeric forms of E2680 were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCVpositive patients were reactive against E2680. By immunizing E2680 in BALB/c mice, both a specific cellular immune response and anti-E2680 IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2680 could be useful to successfully induce strong anti-HCV immunity.

Engineering of N-glycosylation of hepatitis C virus envelope protein E2 enhances T cell responses for DNA immunization

N-linked oligosaccharides are known to be important in modulating immunogenicity of some viral proteins. However, the roles of N-glycans of hepatitis C virus (HCV) E2 envelope glycoprotein in specific cellular immune responses remain elusive. Previous studies showed that the epitopes aa481–500 and aa551–570 of E2 might be important for immunoreactivity and that the binding site of E2 for hCD81 is located at aa480–493 and aa544–551 within the E2 protein. Here, we made plasmids containing genes encoding either wild type or mutant E2 proteins in which N-glycosylation sites (N560NT and N576ST) close to these important regions were mutated separately or in combination. The immunogenicities of wild type E2 and three mutated E2 proteins were analyzed in BALB/c mice using DNA vaccination. The E2-M2 mutant (at N576ST) significantly enhanced (compared to control) E2-specific CTL activity ( P < 0.05), expression of IFN-γ ( P < 0.05) and suppression of tumor growth ( P < 0.05). Also, high IgG2a/IgG1 ratios were elicited in a Th1-type response. These results indicate that engineering of the N-glycosylation site N576ST of HCV E2 protein enhances specific cellular immune responses, providing insights into the development of E2-based DNA vaccines with enhanced immunogenicity.

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.

Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles

Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles

Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-gamma secreting cells, and cytotoxic T lymphocyte assays. Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets. A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope.

Characterization of T‐cell responses against immunodominant epitopes from hepatitis C virus E2 and NS4a proteins

Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti-HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)-alpha therapy and patients who failed to respond to the treatment. Interleukin-2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

Molecular Mechanism of Hepatic Injury in Coinfection with Hepatitis C Virus and HIV

We have previously shown that hepatocytes exposed to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) envelope proteins undergo apoptosis. In this article, we further elucidate the signaling mechanisms that mediate this effect. We found that, in human hepatocellular carcinoma (HepG2) cells, HCV E2 protein and HIV glycoprotein (gp) 120 significantly up-regulated the Fas ligand (FasL) and enhanced the formation of the Fas death-inducing signaling complex downstream of Fas receptor activation. Moreover, after stimulation with HCV E2 and HIV gp120, enhanced expression of caspases 2 and 7 and increased caspase 3 activity were observed. In addition, we showed up-regulation of the proapoptotic molecule Bid and its association with caspase 8 after treatment with these envelope proteins. We also found that HCV E2 and HIV gp120 induced a partial translocation of Bid to the mitochondria, which resulted in the release of cytochrome C and the apoptosis-inducing factor. Thus, the results of this study suggest that FasL and Bid play an important role in HCV and HIV envelope protein-induced apoptosis.

Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors

Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells.

Binding of HCV E2 to CD81 induces RANTES secretion and internalization of CC chemokine receptor 5

Hepatitis C virus (HCV) infection has been shown to be associated with reduced expression of the CC chemokine receptor (CCR) 5, and reduced responsiveness of lymphocytes to chemokines. However, the mechanism by which HCV alters CCR5 expression remains unclear. Here, we investigated whether altered CCR5 expression in hepatitis C results from interactions of CD81 with the HCV E2 protein. Peripheral blood mononuclear cells (PBMC) from HCV-negative individuals were prepared by Ficoll density gradient separation. PBMC subpopulations (CD4+, CD8+ lymphocytes, CD19+ B cells, natural killer (NK) cells and monocyte-derived dendritic cells) were isolated and stimulated with immobilized HCV E2, and changes in CCR5 expression and CC-chemokine secretion were determined. Migration assays were performed using a 5-microm nitrocellulose filter microchamber system according to the manufacturer's recommendations. Exposure of PBMC to HCV E2 induced a dose-dependent release of regulated on activation normal T-cell-expressed and secreted (RANTES), down-regulation of CCR5 expression and intracellular accumulation of CCR5. This effect was blocked by preincubation of PBMC with anti-CD81. RANTES release following exposure to HCV E2 was mainly attributable to CD8+ cells. After exposure to HCV E2 markedly fewer CD8-positive lymphocytes were attracted by RANTES when compared with CD8+ cells that were studied in the absence of HCV E2. Our results suggest that interaction of HCV E2 with CD81 leads to increased RANTES secretion by CD8+ lymphocytes which induces down-regulation of CCR5 surface via receptor internalization resulting in altered lymphocyte migration.