Correlation of the tight junction-like distribution of Claudin-1 to the cellular tropism of hepatitis C virus

Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-alpha treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.     

Effect of cell polarization on hepatitis C virus entry

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.     

Viral kinetics suggests a reconciliation of the disparate observations of the modulation of claudin-1 expression on cells exposed to hepatitis C virus

The tight junction protein claudin-1 (CLDN1) is necessary for hepatitis C virus (HCV) entry into target cells. Recent studies have made disparate observations of the modulation of the expression of CLDN1 on cells following infection by HCV. In one study, the mean CLDN1 expression on cells exposed to HCV declined, whereas in another study HCV infected cells showed increased CLDN1 expression compared to uninfected cells. Consequently, the role of HCV in modulating CLDN1 expression, and hence the frequency of cellular superinfection, remains unclear. Here, we present a possible reconciliation of these disparate observations. We hypothesized that viral kinetics and not necessarily HCV-induced receptor modulation underlies these disparate observations. To test this hypothesis, we constructed a mathematical model of viral kinetics in vitro that mimicked the above experiments. Model predictions provided good fits to the observed evolution of the distribution of CLDN1 expression on cells following exposure to HCV. Cells with higher CLDN1 expression were preferentially infected and outgrown by cells with lower CLDN1 expression, resulting in a decline of the mean CLDN1 expression with time. At the same time, because the susceptibility of cells to infection increased with CLDN1 expression, infected cells tended to have higher CLDN1 expression on average than uninfected cells. Our study thus presents an explanation of the disparate observations of CLDN1 expression following HCV infection and points to the importance of considering viral kinetics in future studies of receptor expression on cells exposed to HCV.     

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry

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.     

Temporal Analysis of Hepatitis C Virus Cell Entry with Occludin Directed Blocking Antibodies

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.     

Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus

Hepatitis C virus (HCV) is a global challenge to public health. Several factors have been proven to be critical for HCV entry, including the newly identified claudin-1 (CLDN1). However, the mechanism of HCV entry is still obscure. Presently, among the 20 members of the claudin family identified in humans so far, CLDN1 has been the only member shown to be necessary for HCV entry. Recently, we discovered that Bel7402, an HCV-permissive cell line, does not express CLDN1 but expresses other members of claudin family. Among these claudins, CLDN9 was able to mediate HCV entry just as efficiently as CLDN1. We then examined if other members of the claudin family could mediate entry. We show that CLDN6 and CLDN9, but not CLDN2, CLDN3, CLDN4, CLDN7, CLDN11, CLDN12, CLDN15, CLDN17, and CLDN23, were able to mediate the entry of HCV into target cells. We found that CLDN6 and CLDN9 are expressed in the liver, the primary site of HCV replication. We also showed that CLDN6 and CLDN9, but not CLDN1, are expressed in peripheral blood mononuclear cells, an additional site of HCV replication. Through sequence comparison and mutagenesis studies, we show that residues N38 and V45 in the first extracellular loop (EL1) of CLDN9 are necessary for HCV entry.     

Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.     

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection

The role of the tight‐junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C‐terminal cytosolic domain in HCV infection. We expressed a series of C‐terminal deletion mutants in Huh‐7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN‐C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans‐Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN‐C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN‐C18. However, in the context of full‐length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN‐C18 in human‐polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.     

Hepatoma Cell Density Promotes Claudin-1 and Scavenger Receptor BI Expression and Hepatitis C Virus Internalization

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.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus, classified in the genus Hepacivirus of the family Flaviviridae. Worldwide, approximately 170 million individuals are persistently infected with HCV, and the majority are at risk of developing chronic liver disease. Hepatocytes in the liver are thought to be the principal reservoir of HCV replication. HCV pseudoparticles (HCVpp) demonstrate a restricted tropism for hepatocyte-derived cells, suggesting that virus-encoded glycoprotein-receptor interactions play an important role in defining HCV tissue specificity.Recent evidence suggests that a number of host cell molecules are important for HCV entry: the tetraspanin CD81; scavenger receptor class B member I (SR-BI) (reviewed in reference 11); members of the tight-junction protein family claudin 1 (CLDN1), CLDN6, and CLDN9 (12, 34, 48, 52); and occludin (OCLN) (2, 33, 40). HCV enters cells via a pH- and clathrin-dependent endocytic pathway; however, the exact role(s) played by each of the host cell molecules in this process is unclear (4, 8, 21, 34, 45).CD81 and SR-BI interact with HCV-encoded E1E2 glycoproteins, suggesting a role in mediating virus attachment to the cell (reviewed in reference 44). In contrast, there is minimal evidence to support direct interaction of CLDN1 or OCLN with HCV particles (12). Evans and colleagues proposed that CLDN1 acts at a late stage in the entry process and facilitates fusion between the virus and host cell membranes (12). We (13, 19) and others (9, 48) have reported that CLDN1 associates with CD81, suggesting a role for CLDN1-CD81 complexes in viral entry. Cukierman et al. recently reported that CLDN1 enrichment at cell-cell contacts may generate specialized membrane domains that promote HCV internalization (9). In this study, we demonstrate that cellular contact modulates SR-BI and CLDN1 expression levels and promotes HCV internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization rates, demonstrating a critical and rate-limiting role for SR-BI in HCV internalization.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.     

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

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.     

Claudin-2 binding peptides,VPDSM and DSMKF,down-regulate claudin-2 expression and anticancer resistance in human lung adenocarcinoma A549 cells

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in spheroid culture models of human lung adenocarcinoma A549 cells. However, there is no chemical which can improve the sensitivity to anticancer drugs. So far, we reported that DFYSP, a short peptide which mimics the second extracellular loop (ECL2) of CLDN2, decreases CLDN2 expression in A549 cells, but the concentration is relatively high. Here, we found that the effects of VPDSM and DSMKF are stronger than that of DFYSP. Both VPDSM and DSMKF decreased the protein levels of CLDN2 without affecting the mRNA levels of CLDN2. The peptide-induced decrease in CLDN2 expression was suppressed by monodansylcadaverine (MDC), a clathrin-dependent endocytosis (CDE) inhibitor, and chloroquine, a lysosome inhibitor. CLDN2 was colocalized with ZO-1, an adapter protein, in tight junctions (TJs) under control conditions, whereas it disappeared from the TJs in the peptide-treated cells. Quartz crystal microbalance assay showed that both peptides can bind to recombinant CLDN2 protein. Both peptides increased permeability to paracellular transport marker lucifer yellow. In three-dimensional spheroid culture models, both peptides enhanced the sensitivity to doxorubicin, a cytotoxic anticancer drug, which was inhibited by MDC. We suggest that VPDSM and DSMKF enhance the chemosensitivity to anticancer drugs in aggregated adenocarcinoma cells mediated by the CDE pathway and lysosomal degradation of CLDN2 in lung adenocarcinoma cells. VPDSM and DSMKF, which mimic the ECL2 of CLDN2, may become novel adjuvant therapeutic drugs for lung adenocarcinoma.     

Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.     

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.     

The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection

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.Hepatitis C virus (HCV) is a small enveloped positive-strand RNA virus that belongs to the Flaviviridae family (20). More than 80% of acute infections become chronic, which eventually progress to cirrhosis and hepatocellular carcinoma (28). HCV infects mainly hepatocytes, but the precise mechanisms of infection are largely unknown (11). The HCV particle consists of a nucleocapsid surrounded by a lipid bilayer in which the two envelope glycoproteins (HCVgp), E1 and E2, are anchored as a heterodimer and play a major role in HCV entry (20). The development of an infectious cell culture model based on the production of infective HCV particles (cell culture-derived HCV particles [HCVcc]) (34) and the generation of HCV pseudotyped retroviral particles (HCVpp) (4) have provided powerful tools to study the HCV cycle. HCV entry is a complex multistep process that requires the presence of several factors. There are multiple pieces of evidence for the involvement of host cell proteins in HCV entry, including glycosaminoglycans, the low-density lipoprotein receptor, scavenger receptor class B type I (SR-BI), and the tetraspanin CD81 (11). Recently, claudin-1, a tight junction (TJ) component, has been identified as a coreceptor required for a late step in HCV entry (13).TJs are major components of cell-cell adhesion complexes and are composed of integral membrane proteins, including occludin and claudins, which associate with actin cytoskeleton-interacting proteins, such as zonula occludens protein 1 (ZO-1) (2). These structures maintain cell polarity, separating apical from basolateral membrane domains, and form a paracellular barrier that allows the selective passage of certain solutes (2). In hepatocytes, TJs seal the bile canaliculi and form the intercellular barrier between bile and blood (12). Recently, we have shown that TJ-associated proteins occludin and claudin-1 disappeared from their normal localization in both HCV-infected and genomic HCV replicon-containing Huh7 cells. Furthermore, TJ function was also altered in these cells (5). In this matter, we have reported an intracellular interaction between E2 and occludin (5). Moreover, it has been reported that claudin-1 and several TJ-associated proteins, such as coxsackievirus and adenovirus receptor (35) and junctional adhesion molecule (JAM) (3), act as virus (co)receptors. Since coxsackievirus entry across epithelial TJs requires occludin (10), we have explored the role of occludin in HCV infection.     

The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus

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.