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A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct-acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384 to 661) that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bNAbs) than the parental form (receptor-binding domain [RBD]). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S, RBD-S, Δ123A7-S, and RBDA7-S; in the last two, 7 cysteines were replaced with alanines. While all four E2-S variant VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bNMAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (NAbs), with three of those animals generating bNAbs against 7 genotypes. Immune serum generated by animals with NAbs mapped to major neutralization epitopes located at residues 412 to 420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease, including cirrhosis and cancer. Broadly neutralizing antibodies that recognize the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here, we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus-like particles that display epitopes recognized by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

Background & Aims: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays., Methods: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps., Results: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones., Conclusions: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories., (Copyright © 2022 AGA Institute. All rights reserved.)

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R 4 Pam 2 Cys and E 8 Pam 2 Cys, to stimulate the production of NAb. We now show that our vaccine with R 4 Pam 2 Cys or E 8 Pam 2 Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E 8 Pam 2 Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.

The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24-peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable. IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins., (Copyright © 2018 Gu et al.)

A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form., Conclusion: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131)., (© 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion., Importance: This study reveals conformational and antigenic differences between the Δ123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure., (Copyright © 2015 Alhammad et al.)

The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384-661). Within E2 are three variable regions located at the N-terminus (HVR1; 384-411), and internally at 460-480 (HVR2) and 570-580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411-428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.

Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.

The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, histidine protonation in the endosome leads to the activation of their membrane fusion function. The HCV (hepatitis C virus) glycoprotein E1-E2 heterodimer mediates membrane fusion within the endosome, but the roles of conserved histidine residues in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidine residues located within E1 and E2. The E1 mutations, H222A/R, H298R and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. A total of five out of six histidine residues located within the E2 RBD (receptor-binding domain) were important for the E2 fold, and their substitution with arginine or alanine caused aberrant heterodimerization and/or CD81 binding. Distinct roles in E1-E2 heterodimerization and in virus entry were identified for His691 and His693 respectively within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH values were enhanced with H445R, indicating that the protonation state of His445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1, indicating a requirement for an endosomal activation trigger in addition to acidic pH.

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.

The three variable regions of hepatitis C virus (HCV) glycoprotein E2 can be removed simultaneously from the E2 ectodomain (residues 384-661) without affecting folding or CD81 binding. In this study, we show that deletion of hypervariable region (HVR) 2 or the intergenotypic variable region (igVR) in the context of the E1E2 polyprotein eliminates formation of heterodimers, reduces CD81 binding and abolishes virus entry. The replication competence of genomic RNA transcribed from the JFH1 infectious HCV clone was not affected by the HVR1, HVR2 or igVR deletions in transfected Huh7.5 cells. However, infectivity of the resultant cell-culture-derived HCV (HCVcc) was abolished by HVR2 or igVR deletions, while deletion of HVR1 led to a 5- to 10-fold reduction in infectivity. Serial passage of cells transfected with genomes lacking HVR1 generated reverted viruses with wild-type levels of infectivity. Sequencing of viral cDNA obtained after full reversion revealed mutations in E1 (I262L) and E2 (N415D) that were present in 35 and 27 % of clones, respectively. Insertion of N415D into HVR1-deleted HCV genomes conferred wild-type levels of infectivity, while I262L increased infectivity by 2.5-fold. These results suggest that HVR2 and the igVR, but not HVR1, are essential for structural integrity and function of the HCV glycoprotein heterodimer.

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

The hepatitis C virus glycoprotein E2 receptor-binding domain is encompassed by amino acids 384 to 661 (E2(661)) and contains two hypervariable sequences, HVR1 and HVR2. E2 sequence comparisons revealed a third variable region, located between residues 570 and 580, that varies widely between genotypes, designated here as igVR, the intergenotypic variable region. A secreted E2(661) glycoprotein with simultaneous deletions of the three variable sequences retained its ability to bind CD81 and conformation-dependent monoclonal antibodies (MAbs) and displayed enhanced binding to a neutralizing MAb directed to E2 immunogenic domain B. Our data provide insights into the E2 structure by suggesting that the three variable regions reside outside a conserved E2 core.

The E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276-286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330-347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.

Background: Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses., Methods/results: The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained., Conclusions: A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G436WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G436WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.

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.

The E1 and E2 glycoproteins of hepatitis C virus form a noncovalently associated heterodimer that mediates viral entry. Glycoprotein E2 comprises a receptor-binding domain (residues 384-661) that is connected to the transmembrane domain (residues 716-746) via a highly conserved sequence containing a hydrophobic heptad repeat (residues 675-699). Alanine- and proline-scanning mutagenesis of the E2 heptad repeat revealed that Leu675, Ser678, Leu689, and Leu692 are important for E1E2 heterodimerization. Furthermore, Pro and Ala substitution of all but one heptad repeat residue (Ser678) blocked the entry of E1E2-HIV-1 pseudotypes into Huh7 cells, irrespective of an effect on heterodimerization. Two conserved prolines (Pro676 and Pro683), occupying consecutive b positions of the heptad, were not required for E1E2 heterodimerization; however, Pro683 was critical for viral entry. Thus, disruption of the predicted alpha-helical structure by proline at position 683 is important for E2 function. The inability of mutants to mediate viral entry was not explained by a loss of receptor binding function, because all mutants were able to interact with a recombinant form of the CD81 large extracellular loop. Chimeras formed between the E1 and E2 ectodomains and the transmembrane domains of flavivirus prM and E glycoproteins, respectively, were able to heterodimerize, although with lower efficiency in comparison with wild type E1E2. The heptad repeat of E2 therefore requires the native transmembrane domain for full heterodimerization and viral entry function. Our data indicate that the membraneproximal heptad repeat of E2 is functionally homologous to the stem of flavivirus E glycoproteins. We propose that E2 has mechanistic features in common with class II fusion proteins.

Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway.

The binding of hepatitis C virus glycoprotein E2 to the large extracellular loop (LEL) of CD81 has been shown to modulate human T-cell and NK cell activity in vitro. Using random mutagenesis of a chimera of maltose-binding protein and LEL residues 113 to 201, we have determined that the E2-binding site on CD81 comprises residues Ile(182), Phe(186), Asn(184), and Leu(162). These findings reveal an E2-binding surface of approximately 806 A(2) and potential target sites for the development of small-molecule inhibitors of E2 binding.