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.     

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication

Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5′ untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3′ portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.MicroRNAs (miRNAs) are a class of noncoding RNA of ∼22-nucleotides in length that can regulate gene expression by either targeting RNA for degradation or suppressing their translation through base pairing to the RNAs (1). Since their discovery in 1993 in Caenorhabditis elegans, miRNAs have been found in many species and are involved in the regulation of proliferation, differentiation, apoptosis, and development (1, 2). Moreover, miRNAs are also critical factors in the development of cancers, neurodegenerative diseases, and infectious diseases (3).MiR-122 is a highly abundant RNA in hepatocytes that regulates lipid metabolism, regeneration, and neoplastic transformation (4–6). In addition, miR-122 is required for the replication of the hepatitis C virus (HCV), a positive-strand RNA virus that infects over 170 million people worldwide (7–9). MiR-122 binds to a conserved sequence in the 5′ untranslated region (UTR) of the HCV RNA to increase the stability of the HCV RNA (10). Silencing of miR-122 can abolish HCV RNA accumulation in non-human primates (11). The expression of human miR-122 in non-hepatic cells can confer the ability to replicate HCV RNA (12). MiR-122 is one of the most critical host factors for HCV replication.We previously reported that the HCV RNA sequence that anneals to miR-122 is recognized by the heterogeneous ribonucleoprotein K (hnRNP K), a multifunctional RNA-binding protein known to be involved in RNA processing, translation, and the replication of several RNA viruses (13–15). In an unbiased screen for proteins from human proteome chips containing over 17,000 proteins, we identified 40 proteins that bind mature miR-122, including hnRNP K. Recombinant hnRNP K recognizes short pyrimidine sequences in miR-122 in vitro and a similar sequence in the HCV 5′ UTR. In hepatocytes endogenous hnRNP K can form a coprecipitable complex with miR-122, whether or not the cells contain replicating HCV. HnRNP K is thus a protein that binds a mature microRNA.     

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.     

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.     

Lack of Adaptation of Chimeric GB Virus B/Hepatitis C Virus in the Marmoset Model: Possible Effects of Bottleneck

Approximately 3% of the world population is chronically infected with hepatitis C virus (HCV). GB virus B (GBV-B), a surrogate model for HCV, causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV. Previously we described a chimeric GBV-B containing an HCV insert from the 5′ noncoding region (NCR) that was adapted for efficient replication in tamarins (Saguinus species). We have also demonstrated that wild-type (WT) GBV-B rapidly adapts for efficient replication in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that the chimeric virus failed to adapt during serial passage in marmosets. The chimeric virus was passaged four times through 24 marmosets. During passage, two marmoset phenotypes were observed: susceptible and partially resistant. Although appearing to adapt in a resistant animal during a prolonged and gradual increase in viremia, the chimeric GBV-B failed to replicate efficiently upon passage to a naïve marmoset. The resistance was specific to the chimeric virus, as the chimeric virus-resistant animals were susceptible to marmoset-adapted WT virus during rechallenge studies. Three isolates of the chimeric virus were sequenced, and 20 nucleotide changes were observed, including eight amino acid changes. Three unique changes were observed in the 5′ NCR chimeric insert, an area that is highly conserved in HCV. We speculate that the failure of the chimeric virus to adapt in marmosets might be due to a bottleneck that occurs at the time of infection of resistant animals, which may lead to a loss of fitness upon serial passage.Worldwide, approximately 170 million people are chronically infected with hepatitis C virus (HCV). The current approved therapy involves the combination of pegylated alpha interferon and ribavirin and has response rates for sustained viral clearance of 42% and 82% for genotypes 1 and 2/3, respectively (15, 29). However, a significant proportion of the population still develops serious disease as a consequence of HCV infection. HCV infection is the leading cause for liver transplantation in the United States (1, 50), and liver cancer due to HCV infection is one of the most rapidly increasing types of cancer in the United States (20).GB virus B (GBV-B) is a hepatotropic virus that causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV (33, 36, 44), and as such, GBV-B represents an important small-primate surrogate model for HCV infections. The history of the GB agent is complex and originates with the inoculation of tamarins with serum obtained from a surgeon with hepatitis (for a review, see reference 3); however there is little doubt that GBV-B is a tamarin virus, despite the fact that it has never been isolated from tamarins a second time. GBV-B has a very narrow host range for tamarins, marmosets, and other closely related New World monkeys (6, 23, 54). The GBV-B model overcomes a number of limitations encountered when working with HCV (3, 22). Due to the limited availability of tamarins, our lab and others (5, 16, 21, 23) initiated GBV-B studies in common marmosets (Callithrix jacchus), a small New World primate closely related to the tamarin (Saguinus sp.). The marmoset and tamarin represent less expensive, more readily available, and smaller animal models than the chimpanzee. While replication in marmosets is typically higher than what is observed in HCV-infected chimpanzees, reproducible infection profiles require some adaptation to this host (5, 54). Although robust replication of HCV in vitro is now possible using specific adapted strains of HCV (derivatives of the JFH1 and H77-S) and the Huh-7.5 cell line (4, 27, 52, 55, 56), the GBV-B primary hepatocyte culture system (2) may be more suitable for some studies, especially those involving specific aspects of the innate immune response and other viral host interactions.The organization of the GBV-B genome is very similar to that of HCV and the GBV-B polyprotein gene encodes 10 proteins analogous to the HCV proteins. The polyprotein of GBV-B has approximately 25 to 30% homology to that of HCV at the amino acid level (33), while the 5′ and 3′ noncoding regions (NCRs) are more divergent (7, 33, 40). The HCV and GBV-B 5′ NCRs are essential for both replication and translation. The structures are similar; however, GBV-B domain I is predicted to fold into two stem-loops (SL), compared to one SL in HCV, and the GBV-B 5′ NCR is longer due mainly to additional SL IIB and IIC that are not present in HCV (Fig. ​(Fig.1A)1A) (40). The 5′ NCR contains the internal ribosomal entry site (IRES), which can directly bind the 40S ribosomal subunit in order to initiate translation of the viral RNA (19, 38). cis RNA elements involved in RNA replication are also located in the HCV 5′ NCR (for a review, see reference 49). In GBV-B, 5′ NCR segments essential for genome replication have recently been identified (53).Open in a separate windowFIG. 1.(A) Predicted structures of the GBV-B (left) and HCV (right) 5′ NCRs. The scissors on GBV-B represent the sequence that was excised and replaced by the HCV insert, which is represented by scissors on the HCV 5′ NCR. The locations of mutations detected during sequencing are boxed: the first box identifies the deletion (ΔC) in a run of cytosines, and the second box identifies the polymorphisms present at two adjacent uracils (C, C/U). (Adapted from reference 40.) (B) Schematic of the GBV-B genome. During passage of GB/III^HC in tamarins, nine mutations were identified in virus from the T2 serum used to inoculate M1, and these nine mutations remained fixed in all marmoset isolates sequenced. Amino acid changes are indicated by dark arrows, silent mutations are indicated by asterisks, and NCR changes are indicated by dotted arrows.The utility of the GBV-B model was increased by the development of infectious cDNA clones that induced hepatitis upon intrahepatic inoculation of tamarins with in vitro-transcribed RNA (7, 31, 45). In order to further increase the use of GBV-B as a model for HCV, chimeras between GBV-B and HCV were constructed (16, 43, 48). In one chimera, a portion of the GBV-B 5′ NCR containing domain III, which is within the IRES functional domain, was replaced by an analogous region of HCV (40-43). The chimeric GB/III^HC retained IRES translational function and supported replication in tamarins (43). In this study, we examined the host range of this chimeric virus during serial passage in marmosets. We found that chimeric GBV-B failed to adapt during passage in marmosets. Marmosets infected with GB/III^HC displayed variable phenotypes ranging from susceptible to resistant, which appear to be due to a polymorphism in the marmoset population that also affects wild-type (WT) GBV-B (54). The failure of chimeric virus to adapt to replication in marmosets with the resistant phenotype was specific to the chimeric virus, and not the WT, and may involve several factors, including reduced replication capacity and the requirement to acquire multiple adaptive mutations. These barriers cumulatively may result in GB/III^HC experiencing a bottleneck in the resistant marmoset host.     

Role of Annexin A2 in the Production of Infectious Hepatitis C Virus Particles

Hepatitis C virus (HCV) is an important human pathogen affecting 170 million chronically infected individuals. In search for cellular proteins involved in HCV replication, we have developed a purification strategy for viral replication complexes and identified annexin A2 (ANXA2) as an associated host factor. ANXA2 colocalized with viral nonstructural proteins in cells harboring genotype 1 or 2 replicons as well as in infected cells. In contrast, we found no obvious colocalization of ANXA2 with replication sites of other positive-strand RNA viruses. The silencing of ANXA2 expression showed no effect on viral RNA replication but resulted in a significant reduction of extra- and intracellular virus titers. Therefore, it seems likely that ANXA2 plays a role in HCV assembly rather than in genome replication or virion release. Colocalization studies with individually expressed HCV nonstructural proteins indicated that NS5A specifically recruits ANXA2, probably by an indirect mechanism. By the deletion of individual NS5A subdomains, we identified domain III (DIII) as being responsible for ANXA2 recruitment. These data identify ANXA2 as a novel host factor contributing, with NS5A, to the formation of infectious HCV particles.Hepatitis C virus (HCV) infections are characterized by a mostly unapparent acute phase leading to persistence in ca. 70% of all infected individuals. Currently, 170 million people suffer from chronic hepatitis C, and they have a high risk to develop severe liver disease. It has been estimated that HCV accounts for 27% of cirrhosis and 25% of hepatocellular carcinoma cases worldwide (2).HCV is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The genome of HCV encompasses a single ∼9,600-nucleotide (nt)-long RNA molecule containing one large open reading frame (ORF) that is flanked by nontranslated regions (NTRs), which are important for viral translation and replication. HCV proteins generated from the polyprotein precursor are cleaved by cellular and viral proteases into at least 10 different products (for a review of polyprotein cleavage and the function of the individual proteins, see reference 4). The structural proteins Core, E1, and E2 are located in the amino-terminal portion of the polyprotein, followed by p7, a hydrophobic peptide that is supposed to be a viroporin, and the nonstructural proteins (NS) NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Only the nonstructural proteins NS3 to NS5B are involved in viral RNA replication. NS3 is a multifunctional protein, consisting of an amino-terminal protease domain required for the processing of the NS3 to NS5B region and a carboxyterminal helicase/nucleoside triphosphatase domain. NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer. The hydrophobic protein NS4B induces vesicular membrane alterations involved in RNA replication. NS5A is a phosphoprotein that seems to play an important role in viral replication and assembly (3, 35, 58). NS5B is the RNA-dependent RNA polymerase of HCV.Positive-strand RNA viruses replicate their RNA in vesicular structures originating from different cellular organelles (36). In the case of HCV, particular membrane alterations have been identified by electron microscopy, designated the membranous web, consisting of accumulations of vesicles primarily derived from the endoplasmic reticulum (17). Important insights into the organization of HCV replication complexes were obtained by the in vitro analysis of viral RNA synthesis in membrane preparations of cells harboring subgenomic HCV replicons, so-called crude replication complexes (CRCs) (1, 20). A current model based on a stoichiometric analysis of CRCs suggests that each vesicular structure contains multiple copies of viral nonstructural proteins and has a connection to the cytoplasm, allowing the constant supply of nucleotides for RNA synthesis (45), presumably analogously to the replication complex of the closely related dengue virus (DV) (64). Viral RNA synthesis in CRCs is highly resistant to proteinases and nucleases (39), and the membranes are detergent resistant at 4°C, resembling features of lipid rafts (54).Several purification techniques have been established to identify relevant HCV host factors by proteomics, based on either the extraction of detergent-resistant membranes (19, 34) or the immunoprecipitation of vesicles (24), revealing different sets of cellular proteins potentially involved in viral replication. In most of these studies, cell lines harboring persistent subgenomic replicons were utilized (33); however, with the availability of a fully permissive cell culture system supporting the complete HCV replication cycle (31, 63, 66), it became evident that viral RNA replication and assembly are closely linked. Recent work revealed an intimate connection of viral replication complexes and assembly sites in close proximity to cytoplasmic lipid droplets (38), with Core and especially NS5A functioning as central regulators by a poorly defined mechanism. NS5A is phosphorylated at multiple serine and threonine residues, binds RNA, and is composed of three domains, which are separated by trypsin-sensitive low-complexity regions (LCS I and II) (59). An N-terminal amphipathic alpha helix tightly associates NS5A with intracellular membranes. Domain I and LCS1 most likely are involved in viral RNA replication, since replication-enhancing mutations primarily mapped to this region (8, 32). The role of domain II is unknown, while domain III recently has been shown to be dispensable for RNA replication but essential for viral particle assembly (3, 35, 58). One of the proposed mechanisms points to a critical interaction with the Core protein, for which phosphorylation in the C-terminal part of domain III of NS5A appears to be required (35). The interaction of Core and NS5A has been proposed to be important for the recruitment of the replication complexes to lipid droplets (3), thereby allowing a coordinated packaging of the newly synthesized RNA.In this study, we identified annexin A2 (also called annexin II, calpactin 1, and ANXA2) as an HCV host factor by a proteomic analysis. ANXA2 belongs to a family of proteins characterized by their Ca²⁺-dependent binding to negatively charged phospholipids. The annexin proteins consist of two principle domains, a variable N-terminal and a conserved C-terminal domain, which harbors the Ca²⁺ and membrane binding sites (for a review, see references 14 and 15). All annexins show cytosolic and membrane localizations. Membrane recruitment probably is regulated by intracellular Ca²⁺ fluctuations, and target membrane selection differs for different annexins.In addition to showing a cytosolic distribution, ANXA2 can associate with the plasma membrane and the membrane of early endosomes. Plasma membrane-associated ANXA2 typically is found in a tight heterotetrameric complex with the S100 protein S100A10 (p11). ANXA2 specifically interacts with phosphatidylinositol(4,5)bisphosphate (PIP2) (22, 48) and binds to membranes enriched in cholesterol, supporting a role in the organization of lipid raft-like membrane microdomains. Due to the direct binding of ANXA2 to F-actin, the protein has been proposed to provide a direct link between cytoskeletal elements and PIP2/cholesterol-rich membrane domains (47).ANXA2 has been implicated in several cellular transport processes, including the internalization and transport of cholesteryl esters, the biogenesis of multivesicular bodies, the recycling of plasma membrane receptors, and the Ca²⁺-induced exocytosis of certain secretory granules (14). Here, we show that ANXA2 is present at HCV replication sites within the membranous web. The recruitment of ANXA2 is mediated by domain III of NS5A and probably is required for efficient virus assembly.     

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.     

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.     

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34, 60, 65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15, 43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8, 16, 59). The internalization of the viral particle occurs by clathrin-mediated endocytosis (5, 40). The fusion of the viral envelope with the membrane of an acidic endosome likely mediates the transfer of the viral genome to the cytosol of the cell (5, 40, 57). However, little is known regarding the pre- and postfusion intracellular transport steps of entering viruses in the endocytic pathway.HCV RNA replication is also associated with cellular membranes. Replication begins with the translation of the genomic RNA of an incoming virus. This leads to the production of viral proteins, which in turn initiate the actual replication of the viral RNA. Mechanisms regulating the transition from the translation of the genomic RNA to its replication are not yet known. All viral proteins are not involved in RNA replication. Studies performed with subgenomic replicons demonstrated that proteins NS3-4A, NS4B, NS5A, and NS5B are necessary and sufficient for replication (6, 27, 37). RNA replication proceeds through the synthesis of a cRNA strand (negative strand), catalyzed by the RNA-dependent RNA polymerase activity of NS5B, which is then used as a template for the synthesis of new positive strands.Electron microscopy studies using a subgenomic replicon model suggested that replication takes place in membrane structures made of small vesicles, referred to as “membranous webs,” which are induced by the virus (26). Membranous webs are detectable not only in cells carrying subgenomic replicons but also in infected cells (50). They appear to be associated with the endoplasmic reticulum (ER) (26). In addition to the membranous webs, a second type of ER-associated replicase that is smaller and more mobile has recently been described (63). Cellular mechanisms leading to these membrane alterations are still poorly understood. In cells replicating and secreting infectious viruses effectively, the situation appears to be even more complex, since replicase components appear to be, at least in part, associated with cytoplasmic lipid droplets (41, 50, 56). This association depends on the capsid protein (41) and may reflect a coupling between replication and assembly. Indeed, HCV assembly and secretion show some similarities with very-low-density lipoprotein (VLDL) maturation and secretion (24, 64).Our knowledge of the cellular membrane mechanisms involved in the HCV life cycle is still limited. The expression of NS4B alone induces membrane alterations that are reminiscent of membranous webs (19). However, cellular factors that participate in this process are still unknown. On the other hand, several cellular proteins potentially involved in the HCV life cycle have been identified through their interactions with viral proteins. For some of these proteins, a functional role in infection was recently confirmed using RNA interference (48). It is very likely that other cellular factors critical to HCV infection have yet to be identified.To gain more insight into cellular mechanisms underlying HCV infection, we made use of brefeldin A (BFA), a macrocyclic lactone of fungal origin that exhibits a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways (30). BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARFs). ARFs are small GTP-binding proteins of the Ras superfamily. They function as regulators of vesicular traffic, actin remodeling, and phospholipid metabolism by recruiting effectors to membranes. BFA does not actually interfere directly with ARF GTPases but rather interferes with their activation by regulators known as guanine nucleotide exchange factors (GEFs) (14, 25). We now report the identification of an ARF GEF as a cellular BFA-sensitive factor that is required for HCV replication.     

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.Recent estimates predict that the prevalence of hepatitis C virus (HCV) infection is approximately 2.2% worldwide, equivalent to about 130 million persons (22). The virus typically establishes a chronic infection that frequently leads to serious liver disease (1), and current models indicate that both morbidity and mortality as a consequence of HCV infection will continue to rise for about the next 20 years (10, 11, 29).HCV is the only assigned species of the Hepacivirus genus within the family Flaviviridae. The virus can be classified into six genetic groups or clades (numbered 1 to 6) and then further separated into subtypes (e.g., 1a, 1b, 2a, 2b, etc.) (53, 55). HCV has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb in length (reviewed in reference 46). Genomic RNA carries a single open reading frame flanked by 5′ and 3′ nontranslated regions, which are important for both replication and translation (19, 20, 34, 47, 56). Viral RNA is translated by the host ribosomal machinery, and the resultant polyprotein is co- and posttranslationally cleaved to generate the mature viral proteins. The structural proteins (core, E1, and E2) and a small hydrophobic polypeptide called p7 are produced by the cellular proteases signal peptidase and signal peptide peptidase (28, 45, 54). Two virus-encoded proteases, the NS2-3 autoprotease and the NS3 serine protease (5, 13, 26), are responsible for maturation of the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). With the exception of NS2, the NS proteins are necessary for genome replication (8, 40) and form replication complexes (RCs), which are located at the endoplasmic reticulum (ER) membrane (14, 24, 52, 57, 59). The functions of all viral constituents of RCs have not been characterized in detail. It is known that NS5B is the RNA-dependent RNA polymerase (6), while NS3 possesses helicase and nucleoside triphosphatase activities in addition to acting as a protease (32, 58). However, the precise roles of the other proteins remain to be firmly established.Expression of NS4B, one of the replicase proteins, generates rearrangements at the ER membrane that have been termed the “membranous web” (14, 24) and “membrane-associated foci” (MAFs) (25). Detection of viral RNA at such foci suggests that NS4B is involved in creating the sites where genome synthesis occurs (18, 24, 59). It is predicted that NS4B has an amphipathic α-helix within its N-terminal region, which is followed by four transmembrane domains (TMDs) in the central portion of the protein (17, 42). As a result, the majority of NS4B is likely to be tightly anchored to membranes, and experimental evidence indicates that it has characteristics consistent with an integral membrane protein (27). It is thought that after membrane association, NS4B rearranges membranes into a network, thereby generating foci which act as a “scaffold” to facilitate RNA replication. The mechanisms engaged in formation of foci are not known but include the notion that the NS4B N terminus can translocate into the ER lumen, resulting in rearrangement of cellular membranes (41, 42). Alternatively, palmitoylation, a lipid modification, might facilitate polymerization of NS4B, in turn promoting formation of RCs on the ER membrane (68).Apart from inducing membranous changes required for replication, NS4B may perform other tasks in HCV RNA synthesis. For example, studies of cell culture adaptive mutations in subgenomic replicons (SGRs) have identified amino acid changes that can stimulate RNA production (39), suggesting that NS4B may exert a regulatory role in determining replication efficiency. In support of a regulatory function, replacement of NS4B sequences in an SGR from strain H77 (a genotype 1a strain) with those from strain Con-1 (a genotype 1b strain) gave higher levels of replication than for a wild-type (wt) strain H77 SGR (7). The corresponding replacement of strain Con-1 NS4B sequences with those from strain H77 reduced the replication efficiency of a Con-1 SGR (7). Moreover, interactions of NS4B with the RC can affect the behavior of other replicase proteins. For example, NS4B is needed for hyperphosphorylation of NS5A (35, 48) and restricts its intracellular movement (30).To try to gain greater insight into the functional organization of the components that constitute RCs, trans-complementation assays using defective and helper SGRs have been established (2, 64). Such studies reveal that the only protein capable of trans-complementation is NS5A, while active replication cannot be restored for replicons harboring deleterious mutations in NS3, NS4B, and NS5B. These data led to the conclusion that functional NS5A may be able to exchange between RCs (2), whereas, by inference, such exchange would not be possible for other HCV replicase proteins. In transient-replication assays, complementation by NS5A also relied on its expression as part of a polyprotein (minimally NS3-NS5A), and production of the protein alone failed to restore replication for an inactive SGR (2). However, in a separate study, stable expression of wt NS5A was capable of complementing a defective replicon (64). Thus, different assay systems can give dissimilar results for complementation by NS5A.In this study, we have created a series of mutations in the NS4B gene of HCV strain JFH1 (31) to explore the function of the protein in the HCV life cycle. We focused our attention on the C-terminal portion of NS4B, downstream from the predicted TMD regions, since it is relatively well conserved and is predicted to lie on the cytosolic side of the ER membrane (15, 42). Our analysis examines the impact of mutations on replication efficiency and the intracellular characteristics of the mutants compared to the behavior of the wt protein. In addition, we have utilized this series of mutants to reassess trans-complementation of NS4B in replication assays. Finally, we also analyze the impact of mutations which do not affect replication on the production of infectious virus to determine whether NS4B plays a role in virus assembly and release.     

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 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.     

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.