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1.  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.
doi:10.1111/cmi.12008
PMCID: PMC3549482  PMID: 22897233
2.  How hepatitis C virus invades hepatocytes: The mystery of viral entry 
Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.
doi:10.3748/wjg.v20.i13.3457
PMCID: PMC3974512  PMID: 24707128
Hepatitis C virus; Virus entry; Hepatocytes; Receptor; Host kinase; Antiviral target
3.  Hepatitis C Virus Induces CD81 and Claudin-1 Endocytosis 
Journal of Virology  2012;86(8):4305-4316.
Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.
doi:10.1128/JVI.06996-11
PMCID: PMC3318669  PMID: 22318146
4.  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.
doi:10.1371/journal.ppat.1001130
PMCID: PMC2951368  PMID: 20949066
5.  Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1 
PLoS ONE  2013;8(5):e64517.
The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
doi:10.1371/journal.pone.0064517
PMCID: PMC3660353  PMID: 23704991
6.  The Tight Junction Proteins Claudin-1, -6, and -9 Are Entry Cofactors for Hepatitis C Virus▿  
Journal of Virology  2008;82(7):3555-3560.
Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81+ cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis.
doi:10.1128/JVI.01977-07
PMCID: PMC2268462  PMID: 18234789
7.  Neutralizing Antibody-Resistant Hepatitis C Virus Cell-to-Cell Transmission ▿ † ‡  
Journal of Virology  2010;85(1):596-605.
Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.
doi:10.1128/JVI.01592-10
PMCID: PMC3014195  PMID: 20962076
8.  Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains 
PLoS Pathogens  2009;5(2):e1000310.
HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.
Author Summary
More than 180 million people are chronically infected by hepatitis C virus (HCV), a leading cause of liver failure and cancer, stimulating the need to fully define the biology of HCV infection for developing novel and effective therapeutics. During the first steps of infection, the virus is taken up and penetrates hepatocytes. HCV entry is thought to be a coordinated multi-step process mediated by specific factors, including CD81, Claudin-1, and the scavenger receptor BI (SR-BI). Whereas the involvement of CD81 and Claudin-1 was demonstrated by rendering susceptible cells that are otherwise refractory, SR-BI complementation assays were lacking, raising questions as to its functions during HCV entry. Here, we identify one hepatoma rat cell line, in which SR-BI complementation assay and targeted mutagenesis could be performed. We therefore demonstrate that SR-BI is an essential HCV entry factor. Our results shed light on SR-BI intracellular domain functions in HCV entry, and, further, emphasize the remarkable capacity of HCV to hijack the lipid transfer function of SR-BI, hence favoring infection.
doi:10.1371/journal.ppat.1000310
PMCID: PMC2636890  PMID: 19229312
9.  Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission 
Journal of Virology  2013;87(15):8282-8293.
Hepatitis C virus (HCV) is believed to initially infect the liver through the basolateral side of hepatocytes, where it engages attachment factors and the coreceptors CD81 and scavenger receptor class B type I (SR-BI). Active transport toward the apical side brings the virus in close proximity of additional entry factors, the tight junction molecules claudin-1 and occludin. HCV is also thought to propagate via cell-to-cell spread, which allows highly efficient virion delivery to neighboring cells. In this study, we compared an adapted HCV genome, clone 2, characterized by superior cell-to cell spread, to its parental genome, J6/JFH-1, with the goal of elucidating the molecular mechanisms of HCV cell-to-cell transmission. We show that CD81 levels on the donor cells influence the efficiency of cell-to-cell spread and CD81 transfer between neighboring cells correlates with the capacity of target cells to become infected. Spread of J6/JFH-1 was blocked by anti-SR-BI antibody or in cells knocked down for SR-BI, suggesting a direct role for this receptor in HCV cell-to-cell transmission. In contrast, clone 2 displayed a significantly reduced dependence on SR-BI for lateral spread. Mutations in E1 and E2 responsible for the enhanced cell-to-cell spread phenotype of clone 2 rendered cell-free virus more susceptible to antibody-mediated neutralization. Our results indicate that although HCV can lose SR-BI dependence for cell-to-cell spread, vulnerability to neutralizing antibodies may limit this evolutionary option in vivo. Combination therapies targeting both the HCV glycoproteins and SR-BI may therefore hold promise for effective control of HCV dissemination.
doi:10.1128/JVI.01102-13
PMCID: PMC3719822  PMID: 23698298
10.  Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts▿  
Journal of Virology  2009;83(11):5477-5484.
Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W30-GLW51-C54-C64. Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.
doi:10.1128/JVI.02262-08
PMCID: PMC2681980  PMID: 19297469
11.  CD81-Receptor Associations — Impact for Hepatitis C Virus Entry and Antiviral Therapies 
Viruses  2014;6(2):875-892.
Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.
doi:10.3390/v6020875
PMCID: PMC3939486  PMID: 24553110
antivirals; claudin-1; kinases; liver; transplantation
12.  Hepatoma Cell Density Promotes Claudin-1 and Scavenger Receptor BI Expression and Hepatitis C Virus Internalization▿  
Journal of Virology  2009;83(23):12407-12414.
Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.
doi:10.1128/JVI.01552-09
PMCID: PMC2786758  PMID: 19776133
13.  Apolipoprotein E on Hepatitis C Virion Facilitates Infection through Interaction with Low Density Lipoprotein Receptor 
Virology  2009;394(1):99-108.
Hepatitis C virus (HCV) infection is a major cause of liver disease. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-I, occludin, and scavenger receptor BI. Here we show that infectious HCV resembles very low density lipoprotein (VLDL) and that entry involves co-receptor function of the low density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that β-VLDL itself or anti-apolipoprotein E (apoE) antibody can block HCV entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. Our results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Disruption of HCV/LDL-R interactions by altering lipoprotein metabolism may therefore represent a focus for future therapy.
doi:10.1016/j.virol.2009.08.037
PMCID: PMC2767442  PMID: 19751943
Hepatitis; HCV; LDL-R; Lipoprotein; ApoE; ApoB; Entry; Receptor
14.  CD81 and Claudin 1 Coreceptor Association: Role in Hepatitis C Virus Entry▿ †  
Journal of Virology  2008;82(10):5007-5020.
Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.
doi:10.1128/JVI.02286-07
PMCID: PMC2346731  PMID: 18337570
15.  HCV entry and neutralizing antibodies: lessons from viral variants 
Future Microbiology  2009;4:511-517.
Summary
Recent data suggest that a strong early broad neutralizing antibody response may contribute to control of hepatitis C virus in the acute phase of infection. However, the majority of individuals fails to clear hepatitis C virus during the first months after contamination and develops chronic infection despite the presence of anti-HCV antibodies. A prerequisite of the understanding of the mechanisms of viral escape from antibody-mediated neutralization has been the identification of various host entry factors mediating the first steps of viral infection: binding and entry of HCV is believed to be a multi-step process involving HCV envelope glycoproteins E1 and E2 as well as several host cell surface molecules such as CD81, scavenger receptor class B type I (SR-BI), members of the claudin family and occludin. In this report, Grove et al. (J Virol 82, 12020-9 (2008)) describe a single mutation in the HCV envelope glycoprotein E2 that alters glycoprotein structure thereby modulating viral interaction with SR-BI and CD81 and increasing sensitivity to neutralizing antibodies. The results of this study highlight the importance of the characterization of the interplay between HCV particles and host cell factors for the understanding of virus neutralization by host immune responses and pathogenesis of HCV infection.
doi:10.2217/fmb.09.34
PMCID: PMC2898794  PMID: 19492962
Hepatitis C virus; adaptive mutation; viral entry; neutralizing antibodies
16.  Hepatitis C virus entry: molecular mechanisms and targets for antiviral therapy 
With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. Using recombinant HCV envelope glycoproteins and HCV pseudotype particles, several cell surface molecules have been identified interacting with HCV during viral binding and entry. These include CD81, highly sulfated heparan sulphate, the low-density lipoprotein receptor, scavenger receptor class B type I and claudin-1. Treatment options for chronic HCV infection are limited and a vaccine to prevent HCV infection is not available. Interfering with HCV entry holds promise for drug design and discovery as the understanding of molecular mechanisms underlying HCV interaction with the host cell is advancing. The complexity of the virus entry process offers several therapeutic targets.
PMCID: PMC3235086  PMID: 19273272
Antiviral Agents; therapeutic use; Hepacivirus; metabolism; physiology; Hepatitis C; drug therapy; Humans; Membrane Fusion; hepatitis C virus; viral entry; entry inhibitor; neutralizing antibodies
17.  Effect of combined siRNA of HCV E2 gene and HCV receptors against HCV 
Virology Journal  2011;8:295.
Background/Aim
Hepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection.
Results
In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number.
Conclusion
From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.
doi:10.1186/1743-422X-8-295
PMCID: PMC3136425  PMID: 21663667
HCV; siRNA; HCV receptors; HCV envelope genes and viral titer
18.  Clinical impact of hepatitis B and C virus envelope glycoproteins 
Chronic infection by either hepatitis B virus (HBV) or hepatitis C virus (HCV) share epidemiological characteristics with risks for development of severe complications such as liver cirrhosis and hepatocellular carcinoma. HBV and HCV also share a high genetic variability. Among highly variable regions, viral genes encoding surface proteins (hepatitis B surface antigen, E1/E2 HCV glycoproteins) play key roles in the stimulation of the host-related immune response and viral entry into hepatocytes. Specific segments of HBV envelope proteins (preS1, “a” determinant) are crucial in the entry process into permissive cells. HCV entry is a complex multistep process involving multiple cell cofactors (glycosaminoglycans, low density lipoprotein receptor, SR-B1, CD81, claudin-1, occludin, EGFR, EphA2) in the interaction with HCV E1/E2 envelope glycoproteins. In vitro both viruses can be controlled by antibody-mediated neutralization targeting viral envelope, also essential in preventing HBV infection in vivo as observed through successful vaccination using HBs antigen. But preventive vaccination and/or therapeutic pressure can influence HBV and HCV variability. For HBV, the patterns of antiviral drug resistance in chronic hepatitis are complex and the original pol/S gene overlap has to be taken into account. Treatment-induced HBV mutations in pol could indeed generate S mutants with subsequent modified antigenicity or increased cancer induction. Variability of HBV and HCV envelope proteins combining high exposure to selective pressures and crucial functional roles require investigation in the context of diagnostic, vaccination and treatment tools. In this editorial a synthesis is performed of HBV and HCV envelope properties at the entry step and as antigenic proteins, and the subsequent clinical impact.
doi:10.3748/wjg.v19.i5.654
PMCID: PMC3574591  PMID: 23429668
Hepatitis B; Hepatitis C; Viral envelope glycoproteins; Clinical outcome
19.  The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection▿  
Journal of Virology  2009;83(16):8012-8020.
The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.
doi:10.1128/JVI.00038-09
PMCID: PMC2715771  PMID: 19515778
20.  Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors 
PLoS Pathogens  2010;6(7):e1000978.
Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.
Author Summary
The hepatitis C virus (HCV) infects only humans and chimpanzees, which has hampered development of suitable animal models. The inability of HCV to penetrate non-human cells is primarily due to inefficient usage of non-human CD81 and occludin. In this study we adapted HCV to mouse CD81. Efficient utilization of mouse CD81 is conferred by a combination of three mutations in the viral glycoproteins. These changes also permit entry via rat or hamster CD81, and lower viral dependence on additional HCV entry factors. Strikingly, mouse CD81 adapted HCV glycoproteins mediate entry into mouse cells in the absence of human entry factors. The adaptive mutations are not resident in viral domains implicated in direct CD81 binding. Nevertheless, they enhance binding to human CD81, increase susceptibility to different neutralizing antibodies and facilitate induction of viral cell fusion by low pH. This suggests that structural changes accompanied by exposure of the CD81 binding site and neutralizing epitopes have “unlocked” the viral envelope protein complex facilitating infection through non-human entry factors. These results highlight mechanisms of HCV receptor usage and tropism. They also demonstrate that HCV can be adapted to using non-human host factors, which may ultimately facilitate the development of small animal models.
doi:10.1371/journal.ppat.1000978
PMCID: PMC2895659  PMID: 20617177
21.  HCV entry receptors as potential targets for siRNA-based inhibition of HCV 
Background
Hepatitis C virus (HCV) is a major health concern with almost 3% of the world's population (350 million individuals) and 10% of the Pakistani population chronically infected with this viral pathogen. The current therapy of interferon-α and ribavirin against HCV has limited efficiency, so alternative options are desperately needed. RNA interference (RNAi), which results in a sequence-specific degradation of HCV RNA has potential as a powerful alternative molecular therapeutic approach. Concerning viral entry, the HCV structural gene E2 is mainly involved in virus attachment to the host cell surface receptors i.e., CD81 tetraspanin, scavenger receptor class B type 1 (SR-B1), low density lipoprotein receptor (LDLR) and claudin1 (CLDN1).
Results
In this report, we studied the relationship of the HCV receptors CD81, LDL, CLDN1 and SR-B1to HCV infection. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was demonstrated by treatment with siRNAs against HCV receptors, which resulted in a significant decrease in HCV viral copy number.
Conclusions
Our data clearly demonstrate that the RNAi-mediated silencing of HCV receptors is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a. These findings will shed further light on the possible role of receptors in inhibition of HCV-3a viral titre through siRNA mediated silencing.
doi:10.1186/1479-0556-9-15
PMCID: PMC3179693  PMID: 21896165
22.  Tight Junction Proteins Claudin-1 and Occludin Control Hepatitis C Virus Entry and Are Downregulated during Infection To Prevent Superinfection ▿ §  
Journal of Virology  2008;83(4):2011-2014.
A tight junction (TJ) protein, claudin-1 (CLDN1), was identified recently as a key factor for hepatitis C virus (HCV) entry. Here, we show that another TJ protein, occludin, is also required for HCV entry. Mutational study of CLDN1 revealed that its tight junctional distribution plays an important role in mediating viral entry. Together, these data support the model in which HCV enters liver cells from the TJ. Interestingly, HCV infection of Huh-7 hepatoma cells downregulated the expression of CLDN1 and occludin, preventing superinfection. The altered TJ protein expression may contribute to the morphological and functional changes observed in HCV-infected hepatocytes.
doi:10.1128/JVI.01888-08
PMCID: PMC2643775  PMID: 19052094
23.  Tupaia CD81, SR-BI, Claudin-1, and Occludin Support Hepatitis C Virus Infection▿  
Journal of Virology  2010;85(6):2793-2802.
Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.
doi:10.1128/JVI.01818-10
PMCID: PMC3067968  PMID: 21177818
24.  Targeting HCV Entry For Development of Therapeutics 
Viruses  2010;2(8):1718-1733.
Recent progress in defining the molecular mechanisms of Hepatitis C Virus (HCV) entry affords the opportunity to exploit new viral and host targets for therapeutic intervention. Entry inhibitors would limit the expansion of the infected cell reservoir, and would complement the many replication inhibitors now under development. The current model for the pathway of entry involves the initial docking of the virus onto the cell surface through interactions of virion envelope and associated low density lipoproteins (LDL) with cell surface glycosaminoglycans and lipoprotein receptors, followed by more specific utilization with other hepatocyte membrane proteins: Scavenger Receptor Class B type 1 (SR-BI), CD81, Claudin 1 (CLDN1) and Occludin (OCLN). The use of blockers of these interactions, e.g. specific antibodies, suggests that inhibition of any one step in the entry pathway can inhibit infection. Despite this knowledge base, the tools for compound screening, HCV pseudoparticles (HCVpp) and cell culture virus (HCVcc), and the ability to adapt them to industrial use are only recently available and as a result drug discovery initiatives are in their infancy. Several therapies aiming at modulating the virus envelope to prevent host cell binding are in early clinical testing. The first test case for blocking a cellular co-receptor is an SR-BI modulator. ITX 5061, an orally active small molecule, targets SR-BI and has shown potent antiviral activity against HCVpp and HCVcc. ITX 5061 has exhibited good safety in previous clinical studies, and is being evaluated in the clinic in chronic HCV patients and patients undergoing liver transplantation. Entry inhibitors promise to be valuable players in the future development of curative therapy against HCV.
doi:10.3390/v2081718
PMCID: PMC3185726  PMID: 21994703
HCV entry inhibitors; SR-BI; HCVpp; therapeutics development
25.  Effect of Cell Polarization on Hepatitis C Virus Entry▿  
Journal of Virology  2007;82(1):461-470.
The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.
doi:10.1128/JVI.01894-07
PMCID: PMC2224355  PMID: 17959672

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