Mouse-Specific Residues of Claudin-1 Limit Hepatitis C Virus Genotype 2a Infection in a Human Hepatocyte Cell Line

Recently, claudin-1 (CLDN1) was identified as a host protein essential for hepatitis C virus (HCV) infection. To evaluate CLDN1 function during virus entry, we searched for hepatocyte cell lines permissive for HCV RNA replication but with limiting endogenous CLDN1 expression, thus permitting receptor complementation assays. These criteria were met by the human hepatoblastoma cell line HuH6, which (i) displays low endogenous CLDN1 levels, (ii) efficiently replicates HCV RNA, and (iii) produces HCV particles with properties similar to those of particles generated in Huh-7.5 cells. Importantly, naïve cells are resistant to HCV genotype 2a infection unless CLDN1 is expressed. Interestingly, complementation of HCV entry by human, rat, or hamster CLDN1 was highly efficient, while mouse CLDN1 (mCLDN1) supported HCV genotype 2a infection with only moderate efficiency. These differences were observed irrespective of whether cells were infected with HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc). Comparatively low entry function of mCLDN1 was observed in HuH6 but not 293T cells, suggesting that species-specific usage of CLDN1 is cell type dependent. Moreover, it was linked to three mouse-specific residues in the second extracellular loop (L152, I155) and the fourth transmembrane helix (V180) of the protein. These determinants could modulate the exposure or affinity of a putative viral binding site on CLDN1 or prevent optimal interaction of CLDN1 with other human cofactors, thus precluding highly efficient infection. HuH6 cells represent a valuable model for analysis of the complete HCV replication cycle in vitro and in particular for analysis of CLDN1 function in HCV cell entry.Hepatitis C virus (HCV) is a liver-tropic plus-strand RNA virus of the family Flaviviridae that has chronically infected about 130 million individuals worldwide. During long-term persistent virus replication, many patients develop significant liver disease which can lead to cirrhosis and hepatocellular carcinoma (54). Current treatment of chronic HCV infection consists of a combination of pegylated alpha interferon and ribavirin. However, this regimen is not curative for all treated patients and is associated with severe side effects (37). Therefore, an improved therapy is needed and numerous HCV-specific drugs targeting viral enzymes are currently being developed (47). These efforts have been slowed down by a lack of small-animal models permissive for HCV replication since HCV infects only humans and chimpanzees. Among small animals, only immunodeficient mice suffering from a transgene-induced disease of endogenous liver cells and repopulated with human primary hepatocytes are susceptible to HCV infection (39).The restricted tropism of HCV likely reflects very specific host factor requirements for entry, RNA replication, assembly, and release of virions. Although HCV RNA replication has been observed in nonhepatic human cells and even nonhuman cells, its efficiency is rather low (2, 11, 59, 67). In addition, so far, efficient production of infectious particles has only been reported with Huh-7 human hepatoma cells, Huh-7-derived cell clones, and LH86 cells (33, 61, 65, 66). Although murine cells sustain HCV RNA replication, they do not produce detectable infectious virions (59). Together, these results suggest that multiple steps of the HCV replication cycle may be blocked or impaired in nonhuman or nonhepatic cells.HCV entry into host cells is complex and involves interactions between viral surface-resident glycoproteins E1 and E2 and multiple host factors. Initial adsorption to the cell surface is likely facilitated by interaction with attachment factors like glycosaminoglycans (4, 31) and lectins (13, 35, 36, 51). Beyond these, additional host proteins have been implicated in HCV entry. Since HCV circulates in the blood associated with lipoproteins (3, 43, 57), it has been postulated that HCV enters hepatocytes via the low-density lipoprotein receptor (LDL-R), and evidence in favor of an involvement of LDL-R has been provided (1, 40, 42, 44). Direct interactions between soluble E2 and scavenger receptor class B type I (SR-BI) (53) and CD81 (49) have been reported, and firm experimental proof has accumulated that these host proteins are essential for HCV infection (5, 6, 16, 26, 28, 33, 41, 61). Finally, more recently, claudin-1 (CLDN1) and occludin, two proteins associated with cellular tight junctions, have been identified as essential host factors for infection (20, 34, 50) and an interaction between E2 and these proteins, as revealed by coimmunoprecipitation assays, was reported (7, 34, 63). Although the precise functions of the individual cellular proteins during HCV infection remain poorly defined, based on kinetic studies with antibodies blocking interactions with SR-BI, CD81, or CLDN1, these factors are likely required subsequent to viral attachment (14, 20, 31, 64). Interestingly, viral resistance to antibodies directed against CLDN1 seems to be slightly delayed compared to resistance to antibodies directed against CD81 and SR-BI (20, 64), suggesting that there may be a sequence of events with the virus encountering first SR-BI and CD81 and subsequently CLDN1. Moreover, in Huh-7 cells, engagement of CD81 by soluble E1/E2 induces Rho GTPase-dependent relocalization of these complexes to areas of cell-to-cell contact, where these colocalized with CLDN1 and occludin (9). Together, these findings are consistent with a model where HCV reaches the basolateral, sinusoid-exposed surface of hepatocytes via the circulation. Upon binding to attachment factors SR-BI and CD81, which are highly expressed in this domain (52), the HCV-receptor complex may be ferried to tight-junction-resident CLDN1 and occludin and finally be endocytosed in a clathrin-dependent fashion (8, 38). Once internalized, the viral genome is ultimately delivered into the cytoplasm through a pH-dependent fusion event (24, 26, 31, 58). Recently, Ploss et al. reported that expression of human SR-BI, CD81, CLDN1, and occludin was sufficient to render human and nonhuman cells permissive for HCV infection (50). These results indicate that these four factors are the minimal cell type-specific set of host proteins essential for HCV entry. Interestingly, HCV seems to usurp at least CD81 and occludin in a very species-specific manner since their murine orthologs permit HCV infection with limited efficiency only (22, 50). Recently, it was shown that expression of mouse SR-BI did not fully restore entry function in Huh-7.5 cells with knockdown of endogenous human SR-BI, suggesting that also SR-BI function in HCV entry is, to some extent, species specific (10).In this study, we have developed a receptor complementation system for CLDN1 that permits the assessment of functional properties of this crucial HCV host factor with cell culture-derived HCV (HCVcc) and a human hepatocyte cell line. This novel model is based on HuH6 cells, which were originally isolated from a male Japanese patient suffering from a hepatoblastoma (15). These cells express little endogenous CLDN1, readily replicate HCV RNA, and produce high numbers of infectious HCVcc particles with properties comparable to those of Huh-7 cell-derived HCV. In addition, we identified three mouse-typic residues of CLDN1 that limit receptor function in HuH6 cells. These results suggest that besides CD81 and occludin, and to a minor degree SR-BI, CLDN1 also contributes to the restricted species tropism of HCV.     

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1_WT, the JFH1_N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1_WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1_T416A and JFH1_I422L more efficiently than the WT virus, while neutralization of JFH1_N417S and JFH1_G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1_N415D, highlighting the importance of the E2 412-423 region in virus entry.Hepatitis C virus (HCV), which belongs to the Flaviviridae family, has a positive-sense single-stranded RNA genome encoding a polyprotein that is cleaved by cellular and viral proteases to yield mature structural and nonstructural proteins. The structural proteins consist of core, E1 and E2, while the nonstructural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (42). The hepatitis C virion comprises the RNA genome surrounded by the structural proteins core (nucleocapsid) and E1 and E2 (envelope glycoproteins). The HCV glycoproteins lie within a lipid envelope surrounding the nucleocapsid and play a major role in HCV entry into host cells (21). The development of retrovirus-based HCV pseudoparticles (HCVpp) (3) and the cell culture infectious clone JFH1 (HCVcc) (61) has provided powerful tools to study HCV entry.HCV entry is initiated by the binding of virus particles to attachment factors which are believed to be glycosaminoglycans (2), low-density lipoprotein receptor (41), and C-type lectins such as DC-SIGN and L-SIGN (12, 37, 38). Upon attachment at least four entry factors are important for particle internalization. These include CD81 (50), SR-BI (53) and the tight junction proteins claudin-1 (15) and occludin (6, 36, 51).CD81, a member of the tetraspanin family, is a cell surface protein with various functions including tissue differentiation, cell-cell adhesion and immune cell maturation (34). It consists of a small and a large extracellular loop (LEL) with four transmembrane domains. Viral entry is dependent on HCV E2 binding to the LEL of CD81 (3, 50). The importance of HCV glycoprotein interaction with CD81 is underlined by the fact that many neutralizing antibodies compete with CD81 and act in a CD81-blocking manner (1, 5, 20, 45).SR-BI is a multiligand receptor expressed on liver cells and on steroidogenic tissue. It binds to high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL) (31). The SR-BI binding site is mapped to the hypervariable region 1 (HVR-1) of HCV E2 (53). SR-BI ligands, such as HDL and oxidized LDL have been found to affect HCV infectivity (4, 14, 58-60). Indeed, HDL has been shown to enhance HCV infection in an SR-BI-dependent manner (4, 14, 58, 59). Antibodies against SR-BI and knockdown of SR-BI in cells result in a significant inhibition of viral infection in both the HCVpp and the HCVcc systems (5, 25, 32).Although clearly involved in entry and immune recognition, the more downstream function(s) of HCV glycoproteins are poorly understood, as their structure has not yet been solved. Nonetheless, mutational analysis and mapping of neutralizing antibody epitopes have delineated several discontinuous regions of E2 that are essential for HCV particle binding and entry (24, 33, 45, 47). One of these is a highly conserved sequence spanning E2 residues 412 to 423 (QLINTNGSWHIN). Several broadly neutralizing monoclonal antibodies (MAbs) bind to this epitope. These include mouse monoclonal antibody (MAb) AP33, rat MAb 3/11, and the human MAbs e137, HCV1, and 95-2 (8, 16, 44, 45, 49). Of these, MAbs AP33, 3/11, and e137 are known to block the binding of E2 to CD81.Cell culture-adaptive mutations within the HCV glycoproteins are valuable for investigating the virus interaction(s) with cellular receptors (18). In the present study, we characterize an asparagine-to-aspartic acid mutation at residue 415 (N415D) in HCV strain JFH1 E2 that arose during the long-term passaging of infected human hepatoma Huh-7 cells. Alongside N415D, we also characterize three adjacent cell culture adaptive mutations reported previously and a novel substitution generated in the present study by propagating virus under MAb AP33 selective pressure to gain further insight into the function of this region of E2 in viral infection.     

Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLN-deficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.Hepatitis C virus (HCV), a member of the family Flaviviridae, is the causative agent of classically defined non-A, non-B hepatitis and is highly prevalent, with approximately 3% of the worldwide population infected (48). HCV infection often results in a chronic, life-long infection that can have severe health consequences, including hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. There is no HCV vaccine available, and the currently employed interferon-based treatment is inadequate as it has severe side effects and is effective only in half of the major genotype-infected individuals (22, 32). Specific anti-HCV inhibitors targeting the viral proteases and polymerase are currently being developed and will likely improve therapeutic options substantially. Undoubtedly, however, the emergence of viral resistance to such inhibitors will be a problem facing future HCV treatment options. As such, developing a spectrum of inhibitors targeting diverse steps in the virus life cycle, including HCV cell entry, is a priority for HCV research. Such inhibitors may be particularly useful following liver transplantation. Although HCV is the leading cause of liver transplants worldwide (10), the usefulness of such procedures is limited by subsequent universal graft reinfection and often accelerated disease progression (21). Even transiently inhibiting graft reinfection with HCV cell entry inhibitors could greatly improve the effectiveness of this procedure. Therefore, a greater understanding of HCV cell entry is required for the development of therapies targeting this stage of the viral life cycle.HCV host cell entry is a complex process that culminates in the clathrin-dependent endocytosis of the virion and low-pH-mediated fusion of viral and cellular lipid membranes in an early endosome (9, 12, 26, 27, 36, 51). The entry process requires the two viral envelope glycoproteins, E1 and E2, and many cellular factors, including glycosaminoglycans (GAGs) (3, 27), lipoproteins, the low-density lipoprotein receptor (LDL-R) (1, 38-40), tetraspanin CD81 (43), scavenger receptor class B type I (SR-BI) (47), and two tight junction proteins, claudin-1 (CLDN1) (17) and occludin (OCLN) (31, 44). The polarized nature of hepatocytes and the tight junction roles of OCLN and CLDN1 suggest an entry pathway similar to that of the group B coxsackieviruses, where the virion initially binds readily accessible factors that then provide a mechanism for migration of the virion into the tight junction region, just prior to internalization (14). Indeed, cellular factors are utilized by the incoming HCV virion in a temporal manner. At least GAGs and LDL-R appear to mediate virion binding (1, 3, 27, 38-40). Conflicting evidence has shown that SR-BI acts as either a binding (11) or postbinding entry factor (53), while CD81 (7, 13, 17, 27) and CLDN1 (17, 29) play postbinding roles in the HCV cell entry process. Although the kinetics of OCLN usage have not been clearly defined, this protein does not appear to play a role in virion binding (6). However, recent data showing that CD81 and CLDN1 may form complexes prior to infection (15, 24, 25, 28, 29, 35, 52) and imaging of the cell entry process (12) may contradict such a model.Human hepatocytes are the major target for HCV infection. While multiple blocks at a number of viral life cycle stages likely exist in other cell types, cell entry is one of the events limiting HCV tropism (45). Although species differences in SR-BI and CLDN1 may exert some influence on this selectivity (11, 23), CD81 and OCLN appear to be largely responsible for the restriction of HCV entry to cells from human and chimpanzee origin (7, 8, 20, 44). In fact, overexpression of the human versions of CD81 and OCLN, along with either mouse or human SR-BI and CLDN1, renders a mouse cell able to support HCV cell entry (44).We sought to provide greater insight into the species-specific restrictions of HCV cell entry and to elucidate the mechanism by which OCLN acts to mediate HCV cell entry. We examined the ability of OCLN proteins from a range of species to mediate HCV cell entry and how this function correlated with the degree of similarity to the human protein. A six-amino-acid portion of the second extracellular loop (EC2) of human OCLN was found to be responsible for the species-specific differences in entry factor function. OCLN proteins that were less functional than the human protein could be rendered fully functional by adding the human residues at these positions. Conversely, the ability of the human OCLN protein to mediate HCV cell entry was impaired by swapping this region with the corresponding sequence from species with less functional OCLN proteins. Comprehensive alanine scanning of the extracellular loops of human OCLN confirmed that the second half of EC2 was most important for the HCV cell entry process. Two cysteine residues that flank this region were found to be essential for HCV cell entry, suggesting that these residues may define a disulfide-linked subdomain of EC2. None of these amino acid changes influenced OCLN expression or localization, implying that they may serve to modulate an interaction with either another host protein or the incoming HCV virion.     

Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

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.Hepatitis C virus (HCV) is a major human pathogen that affects approximately 3% of the global population, leading to cirrhosis and hepatocellular carcinoma in chronically infected individuals (5, 23, 42). Hepatocytes are the major target cells of HCV (11), and entry follows a complex cascade of interactions with several cellular factors (6, 8, 12, 17). Infectious viral particles are associated with lipoproteins and initially attach to target cells via glycosaminoglycans and the low-density lipoprotein receptor (1, 7, 31). These interactions are followed by direct binding of the E2 envelope glycoprotein to the scavenger receptor class B type I (SR-B1) and then to the CD81 tetraspanin (14, 15, 33, 36). Early studies showed that CD81 and SR-B1 were necessary but not sufficient for HCV entry, and claudin-1 was discovered to be a requisite HCV entry cofactor that appears to act at a very late stage of the process (18).Claudin-1 is a member of the claudin protein family that participates in the formation of tight junctions between adjacent cells (25, 30, 37). Tight junctions regulate the paracellular transport of solutes, water, and ions and also generate apical-basal cell polarity (25, 37). In the liver, the apical surfaces of hepatocytes form bile canaliculi, whereas the basolateral surfaces face the underside of the endothelial layer that lines liver sinusoids. Claudin-1 is highly expressed in tight junctions formed by liver hepatocytes as well as on all hepatoma cell lines that are permissive to HCV entry (18, 24, 28). Importantly, nonhepatic cell lines that are engineered to express claudin-1 become permissive to HCV entry (18). Claudin-6 and -9 are two other members of the human claudin family that enable HCV entry into nonpermissive cells (28, 43).The precise role of claudin-1 in HCV entry remains to be determined. A direct interaction between claudins and HCV particles or soluble E2 envelope glycoprotein has not been demonstrated (18; T. Dragic, unpublished data). It is possible that claudin-1 interacts with HCV entry receptors SR-B1 or CD81, thereby modulating their ability to bind to E2. Alternatively, claudin-1 may ferry the receptor-virus complex to fusion-permissive intracellular compartments. Recent studies show that claudin-1 colocalizes with the CD81 tetraspanin at the cell surface of permissive cell lines (22, 34, 41). With respect to nonpermissive cells, one group observed that claudin-1 was predominantly intracellular (41), whereas another reported associations of claudin-1 and CD81 at the cell surface, similar to what is observed in permissive cells (22).Claudins comprise four transmembrane domains along with two extracellular loops and two cytoplasmic domains (19, 20, 25, 30, 37). The first extracellular loop (ECL1) participates in pore formation and influences paracellular charge selectivity (25, 37). It has been shown that the ECL1 of claudin-1 is required for HCV entry (18). All human claudins comprise a highly conserved motif, W₃₀-GLW₅₁-C₅₄-C₆₄, in the crown of ECL1 (25, 37). The exact function of this domain is unknown, and we hypothesized that it is important for HCV entry. The second extracellular loop is required for the holding function and oligomerization of the protein (25). Claudin-1 also comprises various signaling domains and a PDZ binding motif in the intracellular C terminus that binds ZO-1, another major component of tight junctions (30, 32, 37). We further hypothesized that some of these domains may play a role in HCV entry.To understand the role of claudin-1 in HCV infection, we developed a mutagenesis strategy targeting the putative sites for internalization, glycosylation, palmitoylation, and phosphorylation. The functionality of these domains has been described by others (4, 16, 25, 35, 37, 40). We also mutagenized charged and bulky residues in ECL1, including all six residues within the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄. None of the intracellular domains were found to affect HCV entry. However, we identified seven residues in ECL1 that are critical for entry mediated by envelope glycoproteins derived from several HCV subtypes, including all six residues of the conserved motif. These mutants were still expressed at the cell surface and able to form lateral homophilic interactions within the plasma membrane as well as to engage in lateral interactions with CD81. In contrast, they no longer engaged in homophilic trans interactions at cell-cell contacts. We conclude that the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄ of claudin-1 is important for HCV entry into target cells and participates in the formation of cell-cell contacts.     

Role of Scavenger Receptor Class B Type I in Hepatitis C Virus Entry: Kinetics and Molecular Determinants

Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.Hepatitis C virus (HCV) is a global blood-borne pathogen, with 3% of the world''s population chronically infected. Most infections are asymptomatic, yet 60 to 80% become persistent and lead to severe fibrosis and cirrhosis, hepatic failure, or hepatocellular carcinoma (3). Currently available therapies are limited to the administration of pegylated alpha interferon in combination with ribavirin, which are expensive and often unsuccessful, with significant side effects (23, 36). Thus, the development of novel therapeutic approaches against HCV remains a high priority (18, 40, 60). Targeting the early steps of HCV infection may represent one such option, and much effort is being devoted to uncovering the mechanism of viral attachment and entry.The current view is that HCV entry into target cells occurs after attachment to specific cellular receptors via its surface glycoproteins E1 and E2 (27). The molecules to which HCV initially binds might constitute a diverse collection of cellular proteins, carbohydrates, and lipids that concentrate viruses on the cell surface and determine to a large extent which cell types, tissues, and organisms HCV can infect.CD81, claudin 1 (CLDN1), occludin (OCLN), and scavenger receptor class B type I (SR-BI) were previously shown to play essential roles in HCV cell entry (15, 22, 26, 35, 42, 43, 50, 63, 64).Recent reports suggest that CD81 engagement triggers intracellular signaling responses, ultimately leading to actin remodeling and the relocalization of CD81 to tight junctions (TJ) (11). Thus, CD81 may function as a bridge between the initial interaction of the virus with receptors on the basolateral surface of the hepatocyte and the TJ where two of the HCV entry molecules, CLDN1 and OCLN, are located. CD81 acts as a postbinding factor, and the TJ proteins CLDN1 and OCLN seem to be involved in late steps of HCV entry, such as HCV glycoprotein-dependent cell fusion (9, 11, 22). The discovery of TJ proteins as entry factors has added complexity to the model of HCV entry, suggesting parallels with other viruses like coxsackievirus B infection, where an initial interaction of the viral particle with the primary receptor decay-accelerating factor induces the lateral movement of the virus from the luminal surface to TJ, where coxsackievirus B binds coxsackievirus-adenovirus receptor and internalization takes place (17).Much less is known about the specific role of SR-BI in virus entry: neither the specific step of the entry pathway that SR-BI is involved in nor the protein determinants that mediate such processes are known. SR-BI is a lipoprotein receptor of 509 amino acids (aa) with cytoplasmic C- and N-terminal domains separated by a large extracellular domain (1, 13, 14). It is expressed primarily in liver and steroidogenic tissues, where it mediates selective cholesteryl ester uptake from high-density lipoprotein (HDL) and may act as an endocytic receptor (45, 46, 51, 52). SR-BI was originally identified as being a putative receptor for HCV because it binds soluble E2 (sE2) through interactions with E2 hypervariable region 1 (HVR1) (8, 50). RNA interference studies as well as the ability to block both HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) infections with anti SR-BI antibodies have confirmed its involvement in the HCV entry process (7, 8, 15, 26, 33, 63). Intriguingly, lipoproteins were previously shown to modulate HCV infection through SR-BI (12). It was indeed previously demonstrated that two natural ligands of SR-BI, HDL and oxidized low-density lipoprotein, can improve and inhibit HCV entry, respectively (57, 59). Moreover, small-molecule inhibitors of SR-BI-mediated lipid transfer (block of lipid transfer BLT-3 and BLT-4) abrogate the stimulation of HCV infectivity by human serum or HDL, suggesting that the enhancement of viral infection might be dependent on the lipid exchange activity of SR-BI (20, 58).We previously generated high-affinity monoclonal antibodies (MAbs) specific for human SR-BI and showed that they were capable of inhibiting the binding of SR-BI to sE2 and blocking HCVcc infection of human hepatoma cells (15). The HDL-induced enhancement of infection had no impact on the ability of the anti-SR-BI MAbs to block HCV infection, and the antibodies were effective in counteracting HCV infection even in the absence of lipoproteins. These data demonstrated that SR-BI participates in the HCV infection process as an entry receptor by directly interacting with viral glycoproteins. Here we have used one of the anti-SR-BI MAbs to show that SR-BI participates in an early step of HCV infection. By assays of binding of sE2 to SR-BI molecules from different species and to SR-BI mutants, we identified species-specific SR-BI protein residues that are required for sE2 binding. The functional significance of these observations was confirmed by the finding that SR-BI mutants with reduced binding to sE2 were also impaired in their ability to restore the infectivity of an SR-BI-knocked-down Huh-7.5 cell line. Finally, we demonstrated that SR-BI mutants with impaired sE2 binding can still form oligomeric structures and that they can bind the physiological ligand HDL and mediate cholesterol efflux, suggesting that distinct protein determinants are responsible for the interaction with HDL and the HCV particle.     

HLA Class I Subtype-Dependent Expansion of KIR3DS1+ and KIR3DL1+ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection

NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of human immunodeficiency virus type 1 (HIV-1) infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. Here we demonstrate that NK cells expressing the activating receptor KIR3DS1⁺ and, to a lesser extent, the inhibitory receptor KIR3DL1⁺ specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. These data demonstrate for the first time the HLA class I subtype-dependent expansion of specific KIR⁺ NK cells during an acute viral infection in humans.NK cells are cytotoxic effector cells that play a vital role in the innate immune response to viral infections (9, 12, 33). The critical role of NK cells in acute viral infections has been best characterized in acute murine cytomegalovirus (MCMV) infection (14, 28). While several murine lab strains are resistant to MCMV infection, others are highly susceptible. Resistance to MCMV infection was mapped to a gene encoding an activating NK cell receptor, Ly49H, which has been shown to be critical in the early recognition and control of MCMV infection via the direct recognition of a viral product (M157) expressed on infected cells (28). Remarkably, MCMV-infected mice exhibit a dramatic expansion of NK cells during acute infection, but this expansion is restricted to the specific accumulation of Ly49H⁺ NK cells (16). Data from these studies suggest that the antiviral activity of the Ly49H⁺ NK cells is linked to their ability to expand early in infection, prior to the development of adaptive antiviral immunity.While the critical role of Ly49H⁺ NK cells in MCMV infection has been well established, very little is known about the clonal composition of NK cells that expand in human viral infections, and the NK cell receptors that mediate their antiviral activity. Unlike T cells and B cells, the specificity of NK cells is not determined by a single NK cell receptor (8); rather, NK cells express an array of activating and inhibitory receptors that regulate their activity. While the expression of these receptors is stochastic, the random combinations of different receptors on the surface of a given NK cell clone determine its ability to respond to a specific target cell (26, 27). It has been suggested that individual NK cell populations expressing a specific array of receptors may respond differentially to diverse viral infections (7). This has been further supported by epidemiological studies associating the expression of individual activating or inhibitory NK cell receptors in combination with their HLA class I ligands with better or worse disease outcomes in viral infections such as hepatitis C virus (22), human immunodeficiency virus (HIV) (29, 30), human papillomavirus (11), and CMV (7). The functional basis for this protective immunity mediated by NK cells in human viral infections remains largely unknown.Similar to MCMV infection, highly functional NK cells expand rapidly in acute HIV-1 infection, prior to the induction of adaptive immune responses (2). One particular activating killer immunoglobulin-like NK cell receptor (KIR3DS1), in combination with its putative ligand, an HLA-B allele with isoleucine at position 80 (HLA-B Bw480I), has been shown to be associated with slower HIV-1 disease progression (29). We have recently shown that KIR3DS1⁺ NK cells can effectively suppress HIV-1 replication in HLA-B Bw480I⁺ target cells in vitro (1). Furthermore, a subset of inhibitory alleles from the same locus, KIR3DL1, that show high cell surface expression levels have similarly been associated with slower disease progression toward AIDS in the presence of their ligand, HLA-B Bw480I (30). These data suggest that both KIR3DS1⁺ and KIR3DL1⁺ NK cells may play a critical role in the control of natural HIV-1 infection, depending on the interaction with their ligand on infected cells (4). However, the mechanisms underlying their protective role are not understood.Given the critical role of NK cells in acute viral infections and the described expansion of NK cells overall during acute HIV-1 infection (16), we assessed clonal NK cell expansions during acute HIV-1 infection by quantitative PCR and flow cytometric analysis. Here we report an HLA class I subtype-dependent specific expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells during acute HIV-1 infection. These data demonstrate for the first time the impact of the HLA class I ligands on clonal NK cell expansions during an acute human viral infection.     

Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, with approximately 170 million humans chronically infected. Persistent HCV infection often leads to fibrosis, cirrhosis, and hepatocellular carcinoma (27). There is no vaccine against HCV, and the most widely used therapy involves the administration of type I interferon (IFN-α2Α) combined with ribavirin. However, this treatment is often associated with severe adverse effects and is often ineffective (53).HCV is a member of the Flaviviridae family and is the sole member of the genus Hepacivirus (43). HCV is an enveloped virus with a single-strand positive RNA genome that encodes a unique polyprotein of ∼3,000 amino acids (14, 15). A single open reading frame is flanked by untranslated regions (UTRs), the 5′ UTR and 3′ UTR, that contain RNA sequences essential for RNA translation and replication, respectively (17, 18, 26). Translation of the single open reading frame is driven by an internal ribosomal entry site (IRES) sequence residing within the 5′ UTR (26). The resulting polyprotein is processed by cellular and viral proteases into its individual components (reviewed in reference 55). The E1, E2, and core structural proteins are required for particle formation (5, 6) but not for viral RNA replication or translation (7, 40). These processes are mediated by the nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A, and NS5B, which constitute the minimal viral components necessary for efficient viral RNA replication (7, 40).Expression of the viral polyprotein leads to the formation of virus-like particles (VLPs) in HeLa (48) and Huh-7 cells (23). Furthermore, overexpression of core, E1, and E2 is sufficient for the formation of VLPs in insect cells (3, 4). In the context of a viral infection, the viral structural proteins (65), p7 (31, 49, 61), and all of the nonstructural proteins (2, 29, 32, 41, 44, 63, 67) are required for the production of infectious particles, independent of their role in HCV RNA replication. It is not known whether the nonstructural proteins are incorporated into infectious virions.The current model for HCV morphogenesis proposes that the core protein encapsidates the viral genome in areas where endoplasmic reticulum (ER) cisternae are in contact with lipid droplets (47), forming HCV RNA-containing particles that acquire the viral envelope by budding through the ER membrane (59). We along with others showed recently that infectious particle assembly requires microsomal transfer protein (MTP) activity and apolipoprotein B (apoB) (19, 28, 50), suggesting that these two components of the very-low-density lipoprotein (VLDL) biosynthetic machinery are essential for the formation of infectious HCV particles. This idea is supported by the reduced production of infectious HCV particles in cells that express short hairpin RNAs (shRNAs) targeting apolipoprotein E (apoE) (12, 30).HCV RNA displays various density profiles, depending on the stage of the infection at which the sample is obtained (11, 58). The differences in densities and infectivities have been attributed to the presence of host lipoproteins and antibodies bound to the circulating viral particles (24, 58). In patients, HCV immune complexes that have been purified by protein A affinity chromatography contain HCV RNA, core protein, triglycerides, apoB (1), and apoE (51), suggesting that these host factors are components of circulating HCV particles in vivo.Recent studies using infectious molecular clones showed that both host and viral factors can influence the density profile of infectious HCV particles. For example, the mean particle density is reduced by passage of cell culture-grown virus through chimpanzees and chimeric mice whose livers contain human hepatocytes (39). It has also been shown that a point mutation in the viral envelope protein E2 (G451R) increases the mean density and specific infectivity of JFH-1 mutants (70).HCV particles exist as a mixture of infectious and noninfectious particles in ratios ranging from 1:100 to 1:1,000, both in vivo (10) and in cell culture (38, 69). Extracellular infectious HCV particles have a lower average density than their noninfectious counterparts (20, 24, 38). Equilibrium sedimentation analysis indicates that particles with a buoyant density of ∼1.10 to 1.14 g/ml display the highest ratio of infectivity per genome equivalent (GE) both in cell culture (20, 21, 38) and in vivo (8). These results indicate that these samples contain relatively more infectious particles than any other particle population. Interestingly, mutant viruses bearing the G451R E2 mutation display an increased infectivity-HCV RNA ratio only in fractions with a density of ∼1.1 g/ml (21), reinforcing the notion that this population is selectively enriched in infectious particles.The size of infectious HCV particles has been estimated in vivo by filtration (50 to 80 nm) (9, 22) and by rate-zonal centrifugation (54 nm) (51) and in cell culture by calculation of the Stokes radius inferred from the sedimentation velocity of infectious JFH-1 particles (65 to 70 nm) (20). Previous ultrastructural studies using patient-derived material report particles with heterogeneous diameters ranging from 35 to 100 nm (33, 37, 42, 57, 64). Cell culture-derived particles appear to display a diameter within that range (∼55 nm) (65, 68).In this study we exploited the increased growth capacity of a cell culture-adapted virus bearing the G451R mutation in E2 (70) and the enhanced particle production of the hyperpermissive Huh-7 cell subclone Huh-7.5.1 clone 2 (Huh-7.5.1c2) (54) to produce quantities of infectious HCV particles that were sufficient for electron cryomicroscopy (cryoEM) analyses. These studies revealed two major particle populations with diameters of ∼60 and ∼45 nm. The larger-diameter particles were distinguished by the presence of a membrane bilayer, characterized by electron density attributed to the lipid headgroups in its leaflets. Isopycnic ultracentrifugation showed that the ∼60-nm particles are enriched in fractions with a density of ∼1.1 g/ml, where optimal infectivity-HCV RNA ratios are observed. These results indicate that the predominant morphology of the infectious HCV particle is spherical and pleomorphic and surrounded by a membrane envelope.     

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.     

Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine''s anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA''s PPIase activity in the proper assembly and function of the HCV RC.Hepatitis C virus (HCV), of the family Flaviviridae, is an enveloped, positive-stranded RNA virus. Spread mostly by blood-borne transmission, HCV infects more than 170 million people worldwide. The viral genome is composed of a single open reading frame (ORF) plus 5′- and 3′-nontranslated regions. The ORF encodes a large polyprotein that is cleaved by cellular and viral proteases into 10 viral proteins. The structural proteins, including the capsid protein (core), two glycoproteins (E1 and E2), and a small ion channel protein (p7), reside in the N-terminal half of the polyprotein. The rest of the ORF encodes six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3 through NS5B assemble into a replication complex (RC) and are necessary and sufficient for HCV RNA replication in cell culture (8, 42). NS3 is a multifunctional protein with both a serine protease and an RNA helicase activity. The protease activity is responsible for cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (5), and the helicase activity is probably required to unwind the double-stranded RNA intermediates formed during replication (38). NS4A serves as an essential cofactor for the NS3 protease and anchors the NS3 protein to intracellular membranes (25, 36, 39). NS4B induces the formation of a “membranous web” that is probably the site of HCV replication (16). It also contains a GTP-binding motif that is required for replication (17). The web is derived from the endoplasmic reticulum (ER) compartment, although proteins of early-endosome origin have also been found to locate to the web (62). NS5A is a phosphoprotein and an integral component of the viral RC. The precise function of NS5A in replication is still unknown but appears to be regulated by phosphorylation and its interaction with several cellular proteins (19, 22, 24, 51, 52, 59, 63, 67). In addition, it may be involved in the transition from replication and particle formation (4, 45, 64). NS5B is the RNA-dependent RNA polymerase that is responsible for copying the RNA genome of the virus during replication. Several cellular cofactors interact with NS5B and modulate its activity in the context of the viral RC (22, 24, 35, 69, 71).Positive-stranded RNA viruses alter the intracellular membranes of host cells to form an RC in which RNA replication occurs. Modifications include the proliferation and reorganization of certain cellular membranes (1). HCV forms an RC associated with altered cellular membranes (16, 23), and crude RCs (CRCs) that maintain the replicase activity in vitro can be isolated by membrane sedimentation or flotation techniques (2, 3, 18, 27, 37).Cyclosporine is a widely used immunosuppressive and anti-inflammatory drug for organ transplant patients. It functions by forming an inhibitory complex with cyclophilins (CyPs) that inhibits the phosphatase activity of calcineurin, which is important for T-cell activation. In recent years, cyclosporine and its derivatives have been shown to be highly effective in suppressing HCV replication in vitro (44, 49, 53, 68) and in vivo (30). The mechanism of this inhibition is independent of its immunosuppressive function and distinct from that of interferon (IFN) (44, 53, 56, 68).We recently showed that HCV infection in vitro is inhibited when CyPA, a major intracellular target of cyclosporine, is downregulated by RNA interference, and mutations in NS5B that confer cyclosporine-resistant binding to CyPA contribute to the cyclosporine resistance of the replicons harboring these mutations (56, 71). Here we report that CyPA is recruited into the HCV RC together with NS5B in HCV replicon or in HCV-infected cells. Cyclosporine disrupts the association between RC-incorporated NS5B and CyPA and results in an exclusion of the polymerase from the viral RC. We also show that the prolyl-peptidyl isomerase (PPIase) motif of CyPA is essential for HCV replication.     

Pathogenesis of Hepatitis C Virus Infection in Tupaia belangeri

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.Hepatitis C virus (HCV) is a small enveloped virus that causes chronic hepatitis worldwide (32). HCV belongs to the genus Hepacivirus of the family Flaviviridae. Its genome comprises 9.6 kb of single-stranded RNA of positive polarity flanked by highly conserved untranslated regions at both the 5′ and 3′ ends (4, 27, 29). The 5′ untranslated region harbors an internal ribosomal entry site (29) that initiates translation of a single open reading frame encoding a large polyprotein comprising about 3,010 amino acids (35). The encoded polyprotein is co- and posttranslationally processed into 10 individual viral proteins (15).In most cases of human infection, HCV is highly potent and establishes lifelong persistent infection, which progressively leads to chronic hepatitis, liver steatosis, cirrhosis, and hepatocellular carcinoma (9, 16, 21). The most effective therapy for treatment of HCV infection is administration of pegylated interferon combined with ribavirin. However, the combination therapy is an arduous regimen for patients; furthermore, HCV genotype 1b does not respond efficiently (19). The prevailing scientific opinion is that a more viable option than interferon treatment is needed.The chimpanzee is the only validated animal model for in vivo studies of HCV infection, and it is capable of reproducing most aspects of human infection (5, 18, 23, 28, 35, 36). The chimpanzee is also the only validated animal for testing the authenticity and infectivity of cloned viral sequences (8, 14, 35, 36). However, chimpanzees are relatively rare and expensive experimental subjects. Cross-species transmission from infected chimpanzees to other nonhuman primates has been tested but has proven unsuccessful for all species evaluated (1).The tupaia (Tupaia belangeri), a tree shrew, is a small nonprimate mammal indigenous to certain areas of Southeast Asia (6). It is susceptible to infection with a wide range of human-pathogenic viruses, including hepatitis B viruses (13, 20, 31), and appears to be permissive for HCV infection (33, 34). In an initial report, approximately one-third of inoculated animals exhibited acute, transient infection, although none developed the high-titer sustained viremia characteristic of infection in humans and chimpanzees (33). The short duration of follow-up precluded any observation of liver pathology. In addition to the putative in vivo model, cultured primary hepatocytes from tupaias can be infected with HCV, leading to de novo synthesis of HCV RNA (37). These reports strongly support tupaias as a valid model for experimental studies of HCV infection. However, longitudinal analyses evaluating the clinical development and pathology of HCV-infected tupaias have yet to be examined. In the present study, we describe the clinical development and pathology of HCV-infected tupaias over an approximately 3-year time course.     

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.One of the major challenges to overcome in the development of hepatitis C virus (HCV)-directed antivirals is the high propensity of the virus to mutate. This is due to the lack of proofreading capacity of the HCV NS5B RNA-dependent RNA polymerase (RdRp), which replicates the HCV RNA strand with an error rate of 10⁻² to 10⁻³ nucleotide substitutions per site per year (17). The diversity of HCV has been recognized as six phylogenetically distinct groups, referred to as genotypes and, within each genotype, as subtypes (a, b, c, d, etc.) (44). HCV subtype 1b is the most prevalent genotype in the world, and subtype 1a is widely distributed in northern Europe and in the United States; subtypes 2a and 2b are common in North America, Europe, and Japan, and subtype 2c is found predominantly in Northern Italy; HCV genotype 3a is more prevalent in the Far East and has recently increased in Europe and in the United States, possibly due to the spread of the virus through intravenous drug use (11, 17, 18, 44, 46). Of the other genotypes, genotype 4 is common in Africa and to a lesser extent in Europe (11, 39), whereas genotypes 5 and 6 are found predominantly in southern Africa and Southeast Asia, respectively. Despite the availability of a standard of care for the treatment of hepatitis C, a combination of pegylated alpha interferon and ribavirin, many HCV-infected patients cannot be cured because of the frequent failure of the treatment, particularly in patients with genotype 1 and 4 infections, and perhaps also in those with genotype 6 infections (12, 35). In addition, tolerability issues associated with the standard of care lead to discontinuation of therapy in many patients (31). Therefore, major efforts have been made toward developing novel oral therapeutics that target a specific step of the HCV life cycle (45), with particular attention to HCV subtypes 1a and 1b, as they are the most common genotypes underserved by the current standard of care. Subtypes 1a and 1b are estimated to account for 57% and 17% of the HCV-infected patients in the United States (1) versus approximately 11% and 45% in Europe (11), respectively.The development of HCV polymerase nonnucleoside inhibitors (NNIs) has been successfully validated in phase II clinical trials (21, 24, 41). From the extensive screening of NS5B inhibitors that has been performed to date, several chemotypes have emerged as promising scaffolds, namely, the indole, thiophene, benzothiadiazine, and benzofuran analogs. Each of these NNIs targets four different binding pockets of the HCV polymerase, thumb-1 NNI-1 (10), thumb-2 NNI-2 (29, 48), palm-1 NNI-3 (9), and palm-2 NNI-4 (19), respectively. Historically, the screen for novel NS5B inhibitors was limited to representatives of genotype 1b only (3, 28) because the tools to target other genotypes were not yet available (16, 38, 40). Further assessment of these analogs, using enzyme isolates and intergenotypic chimeric replicons derived from clinical isolates, revealed that the genotypic coverage of the NNI-1 and -4 analogs extends beyond genotype 1, unlike the NNI-2 and -3 derivatives that typically inhibit genotype 1 only (16, 38). An additional drawback stems from the lower genetic barrier of the NNI-2 and -3 analogs in genotype 1 (25) and the reduced susceptibility in subtype 1a of the NNI-3 series (7, 16, 34, 38, 43). This effect was mostly attributed to the Y415F polymorphism observed in the NNI-3 binding site in subtype 1a (38).Here we report the 1a/1b subtype profiling of 1,5-benzodiazepine (1,5-BZD) HCV polymerase inhibitors that bind to the NNI-3 site (32, 36) using a panel of enzyme and chimeric replicons derived from clinical isolates, X-ray crystallography, and surface plasmon resonance (SPR) studies and compare these inhibitors to the four classes of NNIs by thoroughly assessing a representative of each nonnucleoside binding site in subtypes 1a and 1b.     

Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal,Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis

Hepatitis B and C viruses (HBV and HCV, respectively) are different and distinct viruses, but there are striking similarities in their disease potential. Infection by either virus can cause chronic hepatitis, liver cirrhosis, and ultimately, liver cancer, despite the fact that no pathogenetic mechanisms are known which are shared by the two viruses. Our recent studies have suggested that replication of either of these viruses upregulates a cellular protein called serine protease inhibitor Kazal (SPIK). Furthermore, the data have shown that cells containing HBV and HCV are more resistant to serine protease-dependent apoptotic death. Since our previous studies have shown that SPIK is an inhibitor of serine protease-dependent apoptosis, it is hypothesized that the upregulation of SPIK caused by HBV and HCV replication leads to cell resistance to apoptosis. The evasion of apoptotic death by infected cells results in persistent viral replication and constant liver inflammation, which leads to gradual accumulation of genetic changes and eventual development of cancer. These findings suggest a possibility by which HBV and HCV, two very different viruses, can share a common mechanism in provoking liver disease and cancer.Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are serious worldwide health problems, with more than 500 million people believed to be chronically infected with at least one of these viruses (36). HBV is a DNA virus belonging to the Hepadnaviridae family (21), while HCV is an RNA virus belonging to the Flaviviridae family (7). Despite the fact that they are two very different viruses, they share a common pathology in the ability to cause chronic hepatitis, liver cirrhosis, and ultimately, hepatocellular carcinoma (HCC) (34). It remains unclear why these two viruses, which are fundamentally so different, can both lead to similar disease states and the development of HCC.Numerous studies suggest that in chronic viral hepatitis, the host''s immune system is unable to clear infected cells (34). The persistent viral replication further stimulates liver inflammation, and prolonged inflammation and viral persistence result in a gradual accumulation of genetic changes which can subsequently lead to transformation and development of HCC (3, 13). It is possible that part of this failure of the host to clear infected cells results from an inability to induce apoptosis in these cells. For example, persistent HBV/HCV infection suppresses cytotoxic-T-lymphocyte (CTL)-induced apoptosis (3, 4). Apoptosis, or programmed cell death, plays a critical role in embryonic development, immune system function, and the overall maintenance of tissue homeostasis in multicellular organisms. It is also important in the host''s control of viral infection (4). The execution of the apoptotic program has traditionally been considered the result of the activation of a family of proteases known as caspases. Caspase-dependent cell apoptosis (CDCA) usually initiates by activating caspases 8 and 10 through proteolysis of their proenzymes, which further activates the executioner caspases, such as caspase 3 and caspase 7, resulting in the degradation of chromosomal DNA and cell death (28, 29). Recent evidence, however, has suggested that apoptotic cell death can also be promoted and triggered by serine proteases in a caspase-independent manner (5, 6, 39). Serine protease-dependent cell apoptosis (SPDCA) differs from CDCA in that serine proteases, not caspases, are critical to the apoptotic process (1, 6, 39). Interestingly, certain viral infections have been shown to induce SPDCA (27, 39).Failure of the immune-mediated removal of malignant cells through apoptosis may be due to the upregulation of apoptosis inhibitors in these cells (12, 18). We recently demonstrated that SPDCA can be inhibited by a small, 79-amino-acid protein called serine protease inhibitor Kazal (SPIK) (22). SPIK, which is also known as SPINK1, TATI (tumor-associated trypsin inhibitor), and PSTI (pancreas secretory trypsin inhibitor) (8, 24, 38), was first discovered in the pancreas as an inhibitor of autoactivation of trypsinogen (9). The expression of SPIK in normal tissue is limited or inactivated outside the pancreas, but expression of SPIK is elevated in numerous cancers, such as colorectal tumors, renal cell carcinoma, gastric carcinoma, and intrahepatic cholangiocarcinoma (ICC) (16, 19, 24, 31, 40, 41). It remains unknown, however, what role SPIK may play in cancer formation and development. Additionally, overexpression of SPIK was also found in HBV/HCV-infected human livers (32), and an even higher level of expression of SPIK was found in HBV/HCV-associated HCC tissue (19, 31). This implies that SPIK may be closely associated with hepatitis virus infection and development of HCC.Here we show direct evidence that HBV/HCV replication does in fact upregulate expression of the apoptosis inhibitor SPIK, resulting in resistance to SPDCA, which could ultimately lead to the development of chronic hepatitis and liver cancer.     

Role of Oxysterol Binding Protein in Hepatitis C Virus infection

Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (ΔPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.Hepatitis C virus (HCV) infection is one of the leading causes of chronic hepatitis. HCV infection is associated with cirrhosis, steatosis, and hepatocellular carcinoma (33). The HCV RNA genome of ∼9.6 kb is translated via an internal ribosome entry site element on the rough endoplasmic reticulum (ER) as a polyprotein precursor of about 3,010 amino acids that is co- and posttranslationally processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins (33). HCV replicates within ribonucleoprotein (RNP) complexes associated with modified ER membranous structures (15). Recent work implicated lipid droplets that emanate from the ER as sites of RNA replication (28, 44). Almost all of the HCV NS proteins along with a variety of cellular factors are associated with the RNP complexes engaged in viral RNA replication (37). It is likely that these NS proteins not only participate in replication process but also are involved in the various steps of virion morphogenesis and assembly. Membrane-associated RNP complexes are generally composed of viral proteins, replicating RNA, host proteins, and altered cellular membranes (1). In this respect, a growing body of evidence implicates the functional role of NS5A in early steps of virion assembly and morphogenesis (3, 27, 45). NS5A is a phosphoprotein that migrates in sodium dodecyl sulfate gels as 56-kDa (basally phosphorylated) and 58-kDa (hyperphosphorylated) forms of proteins. The C-terminal domain III region of NS5A and the phosphorylated residue (Ser⁴⁵⁷) are important for virion maturation (3, 27, 45). NS5A domain III contains the binding site for viral core protein, indicating the possible involvement of NS5A protein in virus assembly (27). NS5A anchors to the ER membrane by an N-terminal hydrophobic α-helix, and this attachment is needed for its key role(s) in viral replication (10). Studies suggest that phosphorylation of NS5A plays a functional role in viral replication (12). The hyperphosphorylated NS5A reduces its interaction with the human vesicle-associated membrane protein-associated protein A (VAP-A) (12). VAP-A binds both NS5A and NS5B (13, 17). These associations are important for RNA replication (13, 17).HCV alters lipid homeostasis to benefit its infectious processes. Host lipids and their synthesis affect viral infectious process (21, 40, 51, 57). HCV RNA replication can be induced by saturated and monounsaturated fatty acids and inhibited by polyunsaturated fatty acids (18, 21). HCV gene expression induces lipogenesis by stimulating the activation of the sterol regulatory element binding proteins, the master regulators of lipid/fatty acid biosynthetic pathways (51). Reagents that interfere with host lipid biosynthetic pathways abrogate viral replication (21, 57). It has been suggested that HCV utilizes the very-low-density lipoprotein (VLDL) secretion pathway for its viral particle release (14, 19). These studies collectively suggest that host lipid metabolism plays a key role in the viral life cycle including replication, virion assembly, and secretion (56).In the present study, we focus on the functional role of oxysterol binding protein (OSBP) that was identified by proteomic analysis as one of the host factors associated with the HCV RNP complexes. OSBP belongs to a family of the OSBP-related proteins. Originally discovered as a major cytosolic receptor for oxidized cholesterols, it undergoes translocation from the cytosolic/vesicular compartment to the Golgi apparatus upon ligand (hydroxycholesterol) binding (38). OSBP also binds to VAP-A via its FFAT motif (53). Golgi apparatus translocation of OSBP is regulated by the pleckstrin homology (PH) domain. This domain also harbors binding sites for phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PI4,5P₂) (25). OSBP and OSBP-related proteins are implicated in cholesterol homeostasis, phospholipid metabolism, vesicular transport, and cell signaling (55). OSBP functions as sterol sensor that regulates the transport of ceramide from the ER to the Golgi apparatus for de novo synthesis of sphingomyelin by coordinated action with ceramide transport protein (CERT) (36). OSBP also functions as a scaffolding protein for two phosphatases (phosphatase 2A/HePTP) (49). This complex regulates the activity of extracellular signal-regulate kinase. This cytosolic 440-kDa complex disassembles by the addition of 25-hydroxycholesterol (25-HC) or depletion of cholesterol, both of which cause OSBP translocation to the Golgi compartment (49). Thus, in addition to its role in intracellular trafficking, OSBP appears to regulate cell signaling. We investigated the functional significance of OSBP association with HCV RNP complexes. RNA interference studies support a functional role of OSBP in virion morphogenesis and release process. The OSBP PH domain deletion mutant (ΔPH) failed to localize to the Golgi apparatus and caused an inhibition of the HCV particle release. Our work described herein also demonstrates that the association of OSBP with NS5A may also contribute to the overall HCV maturation process.     

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.Hepatitis C virus (HCV) infection represents a significant global healthcare burden, and current estimates suggest that a minimum of 3% of the world''s population is chronically infected (4, 19). The virus is responsible for many cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (4, 16, 19). HCV is a positive-stranded RNA virus belonging to the family Flaviviridae. Its ∼9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B reside in the C-terminal half region (6, 34, 44). NS4A, a small 7-kDa protein, functions as a cofactor for NS3 to enhance NS3 enzyme activities such as serine protease and helicase activities. The hydrophobic N-terminal region of NS4A, which is predicted to form a transmembrane α-helix, is responsible for membrane anchorage of the NS3-4A complex (8, 44, 50), and the central region of NS4A is important for the interaction with NS3 (10, 44). A recent study demonstrated the involvement of the C terminus of NS4A in the regulation of NS5A hyperphosphorylation and viral replication (28).The development of HCV replicon technology several years ago accelerated research on viral RNA replication (7, 44). Furthermore, a robust cell culture system for propagation of infectious HCV particles was developed using a viral genome of HCV genotype 2a, JFH-1 strain, enabling us to study every process in the viral life cycle (27, 47, 54). RNA derived from genotype 1a, HCV H77, containing cell-culture adaptive mutations, also produces infectious viruses (52). Using these systems, it has been reported that the HCV genome replicates in a distinct, subcellular replication complex (RC) compartment, which includes NS3-5B and the viral RNA (2, 14, 33). The RC forms in a distinct compartment with high concentrations of viral and cellular components located on detergent-resistant membrane (DRM) structures, possibly a lipid-raft structure (2, 41), which may protect the RC from external proteases and nucleases. Almost all processes in viral replication are dependent on the host cell''s machinery and involve intimate interaction between viral and host proteins. However, the functional roles of host factors interacting with the HCV RC in viral genome replication remain ambiguous.To gain a better understanding of cellular factors that are components of the HCV RC and that function as regulators of viral replication, a comparative proteomic analysis of DRM fractions from HCV replicon and parental cells and subsequent RNA interference (RNAi) silencing of selected genes were performed. We identified creatine kinase B (CKB) as a key factor for the HCV genome replication. CKB catalyzes the reversible transfer of the phosphate group of phosphocreatine (pCr) to ADP to yield ATP and creatine and is known to play important roles in local delivery and cellular compartmentalization of ATP (48, 51). The findings obtained here suggest that recruitment of CKB to the HCV RC, through CKB interaction with NS4A, is essential for maintenance or enhancement of viral replicase activity.     

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus that causes serious liver diseases in humans (31). More than 170 million people worldwide are seropositive for HCV and at risk for developing cirrhosis and hepatocellular carcinoma (50). HCV is a small, enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (31). Its genome encodes a single polyprotein precursor of about 3,000-amino-acid residues that is cleaved co- and posttranslationally by cellular and viral proteases to yield at least 10 mature products (31). The two envelope glycoproteins, E1 and E2, are released from the polyprotein by signal peptidase cleavages. These two proteins assemble as noncovalent heterodimers, which are retained mainly in the endoplasmic reticulum (ER) (36), and they are found as large disulfide-linked oligomers on the surfaces of HCV particles (46). HCV glycoproteins are involved in the entry process, and since they are present on the surfaces of viral particles, these proteins are the targets of neutralizing antibodies (4, 21).E1 and E2 generally contain 4 and 11 N-glycosylation sites, respectively, all of which have been shown to be modified by glycans (19). Despite variability in HCV envelope glycoprotein sequences, the four glycosylation sites of E1 and nine of E2 are highly conserved, suggesting that the glycans associated with these proteins play an essential role in the HCV life cycle (22). Using retroviral particles pseudotyped with genotype 1a (H strain) HCV envelope glycoproteins (HCVpp), recent studies have determined the potential roles played by these glycans in protein folding, HCV entry, and protection against neutralization (14, 19, 22). Indeed, the lack of glycan E1N1, E1N4, E2N8, or E2N10 strongly affects the incorporation of HCV glycoproteins into HCVpp, suggesting that these glycans are important for correct protein folding (19). Furthermore, mutation of glycosylation sites E2N2 or E2N4 alters HCVpp infectivity despite normal incorporation into pseudotyped particles, suggesting a role for the corresponding glycans in viral entry, at least in this model system (19). Finally, glycans at positions E2N1, E2N6, and E2N11 were shown to reduce the sensitivity of HCVpp to antibody neutralization as well as access of the CD81 coreceptor to its binding site on E2, suggesting that glycans also contribute to HCV evasion of the humoral immune response (14, 22).It has recently been proposed that targeting glycans could be a promising approach to inhibiting viral infection (1). Indeed, HCV, as well as several other viruses with highly glycosylated envelope proteins, can be inhibited by carbohydrate binding agents such as cyanovirin-N and pradimicin A (1, 7, 23). Furthermore, resistance against drugs that target glycans is likely to develop and will probably result in mutations at some glycosylation sites (3, 52). However, since glycans associated with viral envelope proteins play an important role in the viral life cycle, adaptation of viruses to the selective pressure of carbohydrate-binding agents will most likely come at a replicative cost to the virus (2).Although the role of HCV glycans has been studied using mutant recombinant HCV envelope glycoproteins incorporated into HCVpp, these particles do not recapitulate all the functions of HCV envelope proteins. Cell culture-derived virus (HCVcc) (32, 49, 55) assembles in an ER-derived compartment in association with very low density lipoproteins (17, 26), whereas HCVpp are assembled in a post-Golgi compartment and are not associated with lipoproteins (44). Importantly, this leads to differences between HCVpp and HCVcc in the oligomerization of the envelope glycoproteins (46). It is also important to note that the carbohydrate composition of viral glycoproteins can differ when the same virus is grown in different cell lines (13). Thus, HCVpp that are produced in 293T cells are not the most appropriate model for glycosylation studies, since HCV tropism is restricted to the liver. Furthermore, differences in envelope protein glycosylation have been observed between HCVpp and HCVcc particles (46). Differences in some HCV envelope protein functions were also observed when the HCVpp and HCVcc systems were compared (28, 29, 42, 43). The development of the HCVcc system provides, therefore, the opportunity to characterize the role of E1/E2-associated glycans in the context of authentic infectious virions. Here, we analyzed the role of E1/E2 glycans by introducing point mutations at N-linked glycosylation sites in the context of the HCVcc system. The effects of these mutations on virus replication, particle secretion, infectivity, and sensitivity to neutralizing antibodies were investigated. Our results demonstrate that several glycans play an important role in HCVcc assembly and/or infectivity and reduce access of neutralizing antibodies to their epitopes.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.