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1.  The role of a hepatitis C virus vaccine: modelling the benefits alongside direct-acting antiviral treatments 
BMC Medicine  2015;13:198.
Hepatitis C virus (HCV) elimination is being seriously considered globally. Current elimination models require a combination of highly effective HCV treatment and harm reduction, but high treatment costs make such strategies prohibitively expensive. Vaccines should play a key role in elimination but their best use alongside treatments is unclear. For three vaccines with different efficacies we used a mathematical model to estimate the additional reduction in HCV prevalence when vaccinating after treatment; and to identify in which settings vaccines could most effectively reduce the number of treatments required to achieve fixed reductions in HCV prevalence among people who inject drugs (PWID).
A deterministic model of HCV transmission among PWID was calibrated for settings with 25, 50 and 75 % chronic HCV prevalence among PWID, stratified by high-risk or low-risk PWID. For vaccines with 30, 60 or 90 % efficacies, different rates of treatment and vaccination were introduced. We compared prevalence reductions achieved by vaccinating after treatment to prevent reinfection and vaccinating independently of treatment history in the community; and by allocating treatments and vaccinations to specific risk groups and proportionally across risk groups.
Vaccinating after treatment was minimally different to vaccinating independently of treatment history, and allocating treatments and vaccinations to specific risk groups was minimally different to allocating them proportionally across risk groups. Vaccines with 30 or 60 % efficacy provided greater additional prevalence reduction per vaccination in a setting with 75 % chronic HCV prevalence among PWID than a 90 % efficacious vaccine in settings with 25 or 50 % chronic HCV prevalence among PWID.
Vaccinating after treatment is an effective and practical method of administration. In settings with high chronic HCV prevalence among PWID, even modest coverage with a low-efficacy vaccine could provide significant additional prevalence reduction beyond treatment alone, and would likely reduce the cost of achieving prevalence reduction targets.
Electronic supplementary material
The online version of this article (doi:10.1186/s12916-015-0440-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4546023  PMID: 26289050
2.  Longitudinal Sequence and Functional Evolution within Glycoprotein E2 in Hepatitis C Virus Genotype 3a Infection 
PLoS ONE  2015;10(5):e0126397.
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.
PMCID: PMC4430534  PMID: 25970466
3.  Editorial on “Broadly neutralizing antibodies abrogate established hepatitis C virus infection” published in Science Translational Medicine on 17th September 2014 
Annals of Translational Medicine  2015;3(Suppl 1):S6.
Hepatitis C virus (HCV) is a blood borne pathogen that causes chronic liver disease and afflicts 170 million people world-wide. While direct acting antivirals now provide a highly effective means to cure those infected with HCV, there is no vaccine to prevent infection. Published in Science Translational Medicine, de Jong et al. [2014] show that highly potent neutralizing antibodies (NAbs) directed to one of the surface glycoproteins of HCV, E2, can not only prevent infection but can also eliminate established infection in experimental animal models of HCV. They provide compelling evidence that for HCV to maintain a chronic infection, it must infect new hepatocytes; infection cannot be sustained in reservoirs of infected cells alone and that E2-specific NAbs are sufficient to cure an infection. In addition, the manuscript further supports the importance of NAbs in preventing, controlling and possibly curing HCV. Thus NAbs are not only essential to the development of prophylactic vaccines but may yet have a role in therapeutic approaches to HCV treatment.
PMCID: PMC4437940  PMID: 26046093
Hepatitis C virus (HCV); neutralizing antibodies (NAbs); vaccine
4.  Challenges to the development of vaccines to hepatitis C virus that elicit neutralizing antibodies 
Despite 20 years of research, a vaccine to prevent hepatitis C virus (HCV) infection has not been developed. A vaccine to prevent HCV will need to induce broadly reactive immunity able to prevent infection by the 7 genetically and antigenically distinct genotypes circulating world-wide. HCV encodes two surface exposed glycoproteins, E1 and E2 that function as a heterodimer to mediate viral entry. Neutralizing antibodies (NAbs) to both E1 and E2 have been described with the major NAb target being E2. The function of E2 is to attach virions to host cells via cell surface receptors that include, but is not limited to, the tetraspanin CD81 and scavenger receptor class B type 1. However, E2 has developed a number of immune evasion strategies to limit the effectiveness of the NAb response and possibly limit the ability of the immune system to generate potent NAbs in natural infection. Hypervariable regions that shield the underlying core domain, subdominant neutralization epitopes and glycan shielding combine to make E2 a difficult target for the immune system. This review summarizes recent information on the role of NAbs to prevent HCV infection, the targets of the NAb response and structural information on glycoprotein E2 in complex with neutralizing antibodies. This new information should provide a framework for the rational design of new vaccine candidates that elicit highly potent broadly reactive NAbs to prevent HCV infection.
PMCID: PMC4080681  PMID: 25071742
glycoprotein E2; neutralizing antibody; CD81; immune evasion; viral entry
5.  Detection of HCV-Specific IFN-γ Responses in HCV Antibody and HCV RNA Negative Injecting Drug Users 
Hepatitis Monthly  2014;14(1):e14678.
Detectable HCV-specific cellular immune responses in HCV antibody and RNA negative people who inject drugs (PWID) raise the question of whether some are resistant to HCV infection. Immune responses from people who have been exposed to hepatitis C virus (HCV) and remain anti-HCV negative are of interest for HCV vaccine development; however, limited research addresses this area.
In a cohort of HCV antibody and RNA negative PWID, we assessed whether the presence of HCV-specific IFN-γ responses or genetic associations provide any evidence of protection from HCV infection.
Patients and Methods:
One hundred and ninety-eight participants were examined longitudinally for clinical, behavioral, social, environmental and genetic characteristics (IFNL3 genotype [formally IL-28B] and HLA type). Sixty-one of the 198 participants were HCV antibody and RNA negative, with 53 able to be examined longitudinally for HCV-specific IFN-γ ELISpot T cell responses.
Ten of the 53 HCV antibody and RNA negative participants had detectable HCV-specific IFN-γ responses at baseline (18%). The magnitude of IFN-γ responses averaged 131 +/- 96 SFC/106 PBMC and the breadth was mean 1 +/- 1 pool positive. The specificity of responses were mainly directed to E2, NS4b and NS5b. Participants with (10) and without (43) HCV-specific IFN-γ responses did not differ in behavioral, clinical or genetic characteristics (P > 0.05). There was a larger proportion sharing needles (with 70%, without 49%, P = 0.320) and a higher incidence of HCV (with 35.1 per 100 py, 95% CI 14.6, 84.4, without 16.0 per 100 py, 95% CI 7.2, 35.6, P = 0.212) in those with IFN-γ responses, although not statistically significant. Half the participants with baseline IFN-γ responses became HCV RNA positive (5/10), with one of these participants spontaneously clearing HCV. The spontaneous clearer had high magnitude and broad Th1 responses, favorable IFNL3 genotype and favorable HLA types.
This study demonstrated the detection of HCV-specific IFN-γ responses in HCV antibody and RNA negative individuals, with a tendency for HCV-specific IFN-γ responses to be associated with HCV exposure. The potential role of HCV-specific IFN-γ responses in those who remained HCV RNA negative is of value for the development of novel HCV therapeutics.
PMCID: PMC3909641  PMID: 24497881
Hepatitis C; Drug Users; Cohort Studies
6.  High Rates of Hepatitis C Virus Reinfection and Spontaneous Clearance of Reinfection in People Who Inject Drugs: A Prospective Cohort Study 
PLoS ONE  2013;8(11):e80216.
Hepatitis C virus reinfection and spontaneous clearance of reinfection were examined in a highly characterised cohort of 188 people who inject drugs over a five-year period. Nine confirmed reinfections and 17 possible reinfections were identified (confirmed reinfections were those genetically distinct from the previous infection and possible reinfections were used to define instances where genetic differences between infections could not be assessed due to lack of availability of hepatitis C virus sequence data). The incidence of confirmed reinfection was 28.8 per 100 person-years (PY), 95%CI: 15.0-55.4; the combined incidence of confirmed and possible reinfection was 24.6 per 100 PY (95%CI: 16.8-36.1). The hazard of hepatitis C reinfection was approximately double that of primary hepatitis C infection; it did not reach statistical significance in confirmed reinfections alone (hazard ratio [HR]: 2.45, 95%CI: 0.87-6.86, p=0.089), but did in confirmed and possible hepatitis C reinfections combined (HR: 1.93, 95%CI: 1.01-3.69, p=0.047) and after adjustment for the number of recent injecting partners and duration of injecting. In multivariable analysis, shorter duration of injection (HR: 0.91; 95%CI: 0.83-0.98; p=0.019) and multiple recent injecting partners (HR: 3.12; 95%CI: 1.08-9.00, p=0.035) were independent predictors of possible and confirmed reinfection. Time to spontaneous clearance was shorter in confirmed reinfection (HR: 5.34, 95%CI: 1.67-17.03, p=0.005) and confirmed and possible reinfection (HR: 3.10, 95%CI: 1.10-8.76, p-value=0.033) than primary infection. Nonetheless, 50% of confirmed reinfections and 41% of confirmed or possible reinfections did not spontaneously clear.
Conclusions: Hepatitis C reinfection and spontaneous clearance of hepatitis C reinfection were observed at high rates, suggesting partial acquired natural immunity to hepatitis C virus. Public health campaigns about the risks of hepatitis C reinfection are required.
PMCID: PMC3820644  PMID: 24244654
7.  Forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of HIV-1 gp41 
Retrovirology  2013;10:44.
The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.
The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER.
The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus.
PMCID: PMC3643854  PMID: 23618462
8.  Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity 
PLoS Pathogens  2013;9(4):e1003218.
The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.
Author Summary
The envelope glycoprotein gp120-gp41 complex of HIV-1 mediates receptor attachment and virus-cell membrane fusion, leading to cellular entry. A shield of asparagine-linked oligosaccharides occludes the gp120-gp41 protein surface and evolution of this glycan shield provides a means for evading circulating neutralizing antibody. Here we examined how conserved structural elements of the glycoprotein complex, in particular the gp120-gp41 association site, retain functionality in the context of glycan shield evolution. This information is important for the evaluation and exploitation of such conserved functional determinants as potential drug and/or vaccine targets. Our data indicate that the loss of either of 2 adjacent glycans in variable region 1 of gp120 leads to changes in local and remote glycan-dependent epitopes and that this is linked to a remodelling of gp120-gp41 interactions in order to maintain a functional gp120-gp41 complex. We propose that this represents a mechanism for the functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of neutralizing antibody selection.
PMCID: PMC3616969  PMID: 23592978
9.  Hepatitis C Virus Nonstructural Protein 5B Is Involved in Virus Morphogenesis 
Journal of Virology  2012;86(9):5080-5088.
The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.
PMCID: PMC3347352  PMID: 22345449
10.  Role of Conserved Cysteine Residues in Hepatitis C Virus Glycoprotein E2 Folding and Function 
Journal of Virology  2012;86(7):3961-3974.
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 [E2661]). 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 E2661, 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, E2661 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 E2661 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.
PMCID: PMC3302498  PMID: 22278231
11.  Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry* 
The Journal of Biological Chemistry  2011;286(37):31984-31992.
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.
PMCID: PMC3173156  PMID: 21768113
Fusion Protein; Glycoprotein; Hepatitis C Virus; Thiol; Virus Entry
12.  The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry 
PLoS Pathogens  2010;6(10):e1001130.
Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor.
Author Summary
Hepatitis C virus (HCV) infection is a major cause of serious liver disease, with approximately 170 million people infected worldwide. Although significant advances have been made in the characterization and development of novel therapeutics to combat HCV infection, there is still a great need for an improved understanding of the HCV lifecycle and potential future targets of antiviral therapy. HCV entry into hepatocytes involves numerous plasma membrane proteins including CD81, scavenger receptor class B type I (SR-BI), claudin-1 and occludin. Although these proteins may comprise the complete set of essential HCV entry factors, the secondary factors that influence the co-ordinated involvement of these proteins in HCV entry remain to be determined. Here we identify the SR-BI partner protein PDZK1 as an indirect regulator of HCV entry. Our results indicate that binding of PDZK1 to the cytoplasmic carboxy-terminus of SR-BI influences the receptor's involvement in HCV entry such that disruption of this interaction may represent a future target of therapeutic intervention.
PMCID: PMC2951368  PMID: 20949066
13.  Claudin Association with CD81 Defines Hepatitis C Virus Entry 
The Journal of Biological Chemistry  2010;285(27):21092-21102.
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
PMCID: PMC2898367  PMID: 20375010
Fluorescence Resonance Energy Transfer (FRET); Receptor Structure-Function; Receptors; Tight Junction; Virus Entry
14.  Identification of a Residue in Hepatitis C Virus E2 Glycoprotein That Determines Scavenger Receptor BI and CD81 Receptor Dependency and Sensitivity to Neutralizing Antibodies ▿  
Journal of Virology  2008;82(24):12020-12029.
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.
PMCID: PMC2593310  PMID: 18829747
15.  Expression and Characterization of a Minimal Hepatitis C Virus Glycoprotein E2 Core Domain That Retains CD81 Binding▿  
Journal of Virology  2007;81(17):9584-9590.
The hepatitis C virus glycoprotein E2 receptor-binding domain is encompassed by amino acids 384 to 661 (E2661) 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 E2661 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.
PMCID: PMC1951388  PMID: 17581991
16.  A Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry 
Journal of Virology  2006;80(16):7844-7853.
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.
PMCID: PMC1563787  PMID: 16873241
17.  Identification of the Hepatitis C Virus E2 Glycoprotein Binding Site on the Large Extracellular Loop of CD81 
Journal of Virology  2002;76(21):11143-11147.
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 Ile182, Phe186, Asn184, and Leu162. These findings reveal an E2-binding surface of approximately 806 Å2 and potential target sites for the development of small-molecule inhibitors of E2 binding.
PMCID: PMC136624  PMID: 12368358
18.  Sequence Conservation and Antigenic Variation of the Structural Proteins of Equine Rhinitis A Virus 
Journal of Virology  2001;75(21):10550-10556.
The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.
PMCID: PMC114636  PMID: 11581430
19.  Functional Analysis of the Disulfide-Bonded Loop/Chain Reversal Region of Human Immunodeficiency Virus Type 1 gp41 Reveals a Critical Role in gp120-gp41 Association 
Journal of Virology  2001;75(14):6635-6644.
Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614–6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.
PMCID: PMC114387  PMID: 11413331
20.  In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface 
Cellular Microbiology  2012;14(12):1892-1903.
Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62–66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCVglycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.
PMCID: PMC3549482  PMID: 22897233

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