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.     

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.     

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.     

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 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.     

Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.Hepatitis C virus (HCV) is the major causative agent of non-A/non-B hepatitis (26). More than 170 million people, or 3% of the population in the world, are infected with HCV (29). It establishes chronic infection in at least 85% of infected individuals and is associated with liver cirrhosis and hepatocellular carcinoma. Current treatment, which combines polyethylene glycol-interferon (PEG-IFN) and ribavirin, is ineffective in 22% of patients with non-genotype 1 and in 45% of patients with genotype 1 HCV (1, 16, 23, 55). Therefore, identification of new targets for HCV therapy is an important issue, and cellular genes involved in the HCV life cycle may serve as good candidates.HCV is a positive-strand RNA virus and the only known member of Hepacivirus genus in the family Flaviviridae. Its genome has a length of about 9,600 nucleotides coding for a single polyprotein. The long polyprotein is further processed into at least 10 different products, including four structural proteins (core, E1, E2, and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Nonstructural proteins NS3-NS5B are components of the membrane-associated HCV replication complex (8, 13, 36, 45). NS3 is a bifunctional protein containing an N-terminal protease domain and a C-terminal helicase/NTPase domain, and NS4A serves as a cofactor for NS3 protease. NS4B protein is known to induce intracellular membrane changes that probably serve as the site for viral RNA replication (8). NS5A is required for RNA replication, but little is known about its function. NS5B is the RNA-dependent RNA polymerase (reviewed in reference 47).NS5A is phosphorylated on multiple serine and threonine residues and exists in basal phosphorylated (p56) and hyperphosphorylated (p58) forms (49). Increasing evidence suggests that the regulation of NS5A phosphorylation is important for HCV RNA replication. Adaptive mutations or kinase inhibitors, which reduce NS5A hyperphosphorylation, increased the replication of an HCV replicon in cell culture (HCVcc) systems (2, 4, 38). However, when an adaptive replicon with reduced p58 was further treated with the same kinase inhibitor or introduced with a second adaptive mutation, RNA replication was completely blocked (32, 38). Furthermore, the mutations that reduce NS5A hyperphosphorylation and promote RNA replication in cell culture, paradoxically, prevented productive replication in the chimpanzee model (6). These results imply that the tight control of the p58/p56 ratio is important for HCV replication. The detailed mechanism is still not clear, but a clue was provided by the finding of differential association of NS5A phospho-forms with the host vesicle-associated membrane protein-associated protein A (VAP-A) protein, which is an essential molecule for HCV replicase (9, 12). On the other hand, NS5A phosphorylation was recently found to regulate the production of infectious virus (34, 50). Alanine substitutions in the C-terminal domain III of NS5A impaired NS5A phosphorylation, leading to a decrease in NS5A-core protein interaction, disturbance of subcellular localization of NS5A, and disruption of virion production (3, 34, 50). In summary, phosphorylation on NS5A is not only important for HCV RNA replication but also critical for infectious virus production.Since the phosphorylation state of NS5A is correlated with HCV RNA replication and virion production, cellular kinases responsible for NS5A phosphorylation may serve as good candidates for drug targets. Several kinases have been shown to target NS5A in vitro, including casein kinase I (CKI), CKII, MEK1, MKK6, MKK7, AKT, and p70S6K (7, 24). Among these proteins, CKI and CKII are better characterized for NS5A phosphorylation. CKIα has been identified as the target of kinase inhibitors which decrease the hyperphosphorylation of NS5A and was further confirmed as a direct kinase of NS5A (41, 42). CKI requires prephosphorylation of residues near the predicted phosphorylation site in NS5A for effective modification, suggesting that other kinases are also involved in this process (42). CKII has been shown to bind to the C-terminal domain of NS5A and phosphorylate NS5A in vitro (24). Inhibition of CKII with chemical compounds or small interfering RNA (siRNA) did not significantly affect HCV RNA replication but severely disrupted virus production (50).In this study, using lentivirus-based RNA interference (RNAi) screening, we identified a serine/threonine kinase, Polo-like kinase 1 (Plk1), which is involved in HCV replication. Expression of short hairpin RNAs (shRNAs) targeting Plk1 decreased HCV replication and virus production. Moreover, silencing of Plk1 decreased the hyperphosphorylated form of NS5A. In cells treated with a Plk1-specific kinase inhibitor, HCV replication and NS5A hyperphosphorylation were significantly reduced, indicating that Plk1 kinase activity is required for this process. Further studies showed that Plk1 was coimmunoprecipitated and partially colocalized with NS5A, suggesting NS5A as a possible substrate for Plk1. Finally, NS5A is hyperphosphorylated by Plk1 in vitro, supporting the proposition that Plk1 regulates HCV replication through hyperphosphorylation of NS5A.     

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.     

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.     

GBF1, a Guanine Nucleotide Exchange Factor for Arf,Is Crucial for Coxsackievirus B3 RNA Replication

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.Enteroviruses are small, nonenveloped, positive-stranded RNA viruses that include many important pathogens, such as poliovirus (PV), coxsackievirus, echovirus, and human rhinovirus. Following virus entry and uncoating, the 7.5-kb enteroviral RNA genome is directly translated into a large polyprotein. This polyprotein is proteolytically processed by the virus-encoded proteases 2A^pro, 3C^pro, and 3CD^pro into the structural P1 region proteins and the nonstructural P2 and P3 region proteins that are involved in viral RNA replication.All RNA viruses with a positive-stranded genome induce the remodeling of cellular membranes to create a scaffold for genomic RNA replication. The organelle origin and morphology of these membranous replication sites, however, appear to vary for different viruses. Enteroviruses replicate their RNA genomes in nucleoprotein complexes that are associated with small vesicular membrane structures (6). The enteroviral proteins 2B, 2C, and 3A have been implicated in vesicle formation (4, 6, 27), but the mechanism and pathway of membrane reorganization are poorly understood. There are strong indications that these vesicular membranous structures, which are referred to here as “vesicles,” are derived from the early secretory pathway. Vesicles produced in PV-infected cells may form at the endoplasmic reticulum (ER) by the cellular COP-II budding machinery and may therefore share components with the membranous vesicles mediating ER-to-Golgi network transport (26). Further support for the involvement of the secretory pathway stems from the observation that brefeldin A (BFA), a well-known inhibitor of ER-to-Golgi network transport, completely inhibits enteroviral RNA replication (17, 20). In addition, the autophagocytic pathway appears to contribute to the formation of the membrane vesicles, many of which exhibit a double-membrane morphology characteristic of autophagosomes (18, 27). The utilization of individual components or reactions from different membrane metabolic pathways, rather than subversion of an entire pathway in toto, may represent a common strategy for building viral replication machinery.BFA inhibits activation of the small monomeric GTPase ADP ribosylation factor 1 (Arf1), a major regulator of intracellular protein transport (2). Arf1 cycles between an inactive, GDP-bound, cytosolic state and an active, GTP-bound, membrane-associated state, and this cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (13). BFA blocks the activities of the large GEFs GBF1, BIG1, and BIG2 by stabilizing an intermediate, abortive complex with inactive Arf1 (23), thus efficiently preventing activation of Arf1 and eventually formation of transport intermediates.Not only the fact that BFA blocks enteroviral replication suggests a role for Arf1 and/or its large GEFs in this process; recently, it was shown that Arf1 accumulates on membranes during PV infection (3). Arf1 translocation to membranes can be induced independently by enterovirus protein 3A or 3CD in vitro (5), but the underlying mechanisms seem to differ; the 3A protein specifically triggers the recruitment of GBF1 to membranes, most likely through a direct interaction with this GEF (32, 33), whereas 3CD recruits BIG1 and BIG2 to membranes (3). Here, we report the involvement of Arf1 and its large BFA-sensitive GEFs in coxsackievirus B3 (CVB3) replication.     

A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.The compact genomes of viruses relative to those of other infectious agents restrict their ability to encode all proteins required to complete their replication cycles. To circumvent this limitation, viruses often utilize cellular factors or processes to complete essential steps in replication. One group of cellular proteins frequently targeted by viruses are cellular chaperones, which include a diverse set of heat shock proteins (hsps) that normally facilitate cellular protein translation, folding, trafficking, and degradation (18, 64). The connection between viruses and cellular chaperones was originally identified in bacteria, where the Escherichia coli hsp40 and hsp70 homologues, encoded by dnaJ and dnaK, respectively, were identified as bacterial genes essential for bacteriophage λ DNA replication (62). Research over the past 30 years has further revealed the importance of cellular chaperones in viral replication, such that the list of virus-hsp connections is now quite extensive and includes viruses from numerous families with diverse genome structures (4, 6, 7, 16, 19, 20, 23, 25, 40, 41, 44, 51, 54, 60). These studies have demonstrated the importance of cellular chaperones in multiple steps of the viral life cycle, including entry, viral protein translation, genome replication, encapsidation, and virion release. However, the list of virus-hsp connections is likely incomplete. Further studies to explore this particular host-pathogen interaction will shed light on virus replication mechanisms and pathogenesis, and potentially highlight targets for novel antiviral agents.To study the role of cellular chaperones in the genome replication of positive-sense RNA viruses, we use flock house virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae family. The FHV life cycle shares many common features with other positive-sense RNA viruses, including the membrane-specific targeting and assembly of functional RNA replication complexes (37, 38), the exploitation of various cellular processes and host factors for viral replication (5, 23, 60), and the induction of large-scale membrane rearrangements (24, 28, 38, 39). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the structural capsid protein precursor, respectively (1). During active genome replication, FHV produces a subgenomic RNA3 (0.4 kb), which encodes the RNA interference inhibitor protein B2 (12, 29, 32). These viral characteristics make FHV an excellent model system to study many aspects of positive-sense RNA virus biology.In addition to the benefits of a simple genome, FHV is able to establish robust RNA replication in a wide variety of genetically tractable eukaryotic hosts, including Drosophila melanogaster (38), Caenorhabditis elegans (32), and Saccharomyces cerevisiae (46). The budding yeast S. cerevisiae has been an exceptionally useful model host to study the mechanisms of viral RNA replication complex assembly and function with FHV (31, 37, 39, 45, 53, 55, 56, 60) as well as other positive-sense RNA viruses (11). The facile genetics of S. cerevisiae, along with the vast array of well-defined cellular and molecular tools and techniques, make it an ideal eukaryotic host for the identification of cellular factors required for positive-sense RNA virus replication. Furthermore, readily available yeast libraries with deletions and regulated expression of individual proteins have led to the completion of several high-throughput screens to provide a global survey of host factors that impact virus replication (26, 42, 52). An alternative approach with these yeast libraries that reduces the inherently high false-negative rates associated with high-throughput screens is to focus on a select set of host genes associated with a particular cellular pathway, process, or location previously implicated in virus replication.We have utilized such a targeted approach and focused on examining the impact of cytosolic chaperones on FHV RNA replication. Previously, we have shown that the cellular chaperone hsp90 facilitates protein A synthesis in Drosophila cells (5, 23), and the hsp40 encoded by the yeast YDJ1 gene facilitates FHV RNA replication in yeast, in part through effects on both protein A accumulation and function (60). In this report, we further extend these observations by examining FHV RNA accumulation in a panel of yeast strains with deletions of known or hypothesized cytosolic chaperones. We demonstrate that cytosolic chaperones can have either suppressive or enhancing effects on FHV RNA accumulation. In particular, related hsp70 members encoded by the SSA and SSB yeast chaperone families have marked and dramatically divergent effects on both genomic and subgenomic RNA accumulation and viral polymerase synthesis. These results highlight the complexities of the host-pathogen interactions that influence positive-sense RNA virus replication and identify the hsp70 family of cytosolic chaperones as key regulators of FHV replication.     

Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV_TCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV_TCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV_TCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10⁶ tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV_TCP may prove valuable for gene delivery or vaccination approaches.Hepatitis C virus (HCV) is an enveloped plus-strand RNA virus of the genus Hepacivirus within the family Flaviviridae (34). The HCV genome is approximately 9.6 kb in length and consists of a single open reading frame encoding a polyprotein of ca. 3,000 amino acids and nontranslated regions (NTRs) located at the 5′ and 3′ termini. These NTRs are highly structured RNA segments encompassing critical cis-active RNA elements essential for genome replication and RNA translation (31). Viral proteins are expressed in a cap-independent manner by means of an internal ribosome entry site (IRES) located in the 5′ NTR. Co- and posttranslational cleavages liberate 10 viral proteins: core; envelope protein 1 (E1) and E2, representing the structural proteins that constitute the virion; a small membrane-associated ion channel protein designated p7 that is essential for virus assembly (16, 22, 43, 57); and six nonstructural (NS) proteins (NSs 2, 3, 4A, 4B, 5A, and 5B). HCV proteins NS3 to NS5B are both necessary and sufficient to establish membrane-bound replication complexes catalyzing RNA replication (13, 36). More recent data indicate that the NS2 protease that catalyzes cleavage at the NS2-NS3 site in addition participates in assembly and release of infectious viruses (22). Finally, ribosomal frame-shifting and internal translation initiation yield a group of additional proteins designated ARFP (alternative reading frame protein) or core+1 proteins. However, their function for the HCV replication cycle is currently not known.One hallmark of HCV is its high propensity to establish a persistent infection, which frequently causes progressive morbidity ranging from hepatic fibrosis to cirrhosis and hepatocellular carcinoma (20). Despite considerable progress in the treatment of HCV infection, the currently available therapy (a combination of pegylated interferon alpha with ribavirin) is not well tolerated and is efficacious in only ca. 50% of patients infected with the most prevalent genotype 1 (38). Therapeutic or prophylactic vaccines are not available. For these reasons and with currently ca. 170 million persistently infected individuals, HCV infection represents a considerable global health problem necessitating pertinent basic and applied research efforts.In recent years three major advances enabled analysis of the HCV replication cycle in tissue culture. First, Lohmann and colleagues developed subgenomic HCV replicons (36). These autonomously replicating RNA molecules carry all the genetic elements necessary for self-replication (the NTRs and NS3 to NS5B), including a selectable marker or a reporter gene in place of the viral structural proteins, and an internal IRES for expression of the HCV replicase genes (reviewed in reference 45). Second, HCV pseudotype particles, i.e., retroviral particles surrounded by an envelope carrying HCV E1-E2 complexes in place of their cognate envelope proteins, were established (3, 21). As these particles carry functional HCV glycoprotein complexes on their surface, HCV pseudotype particles have been instrumental for the analysis of E1-E2 receptor interactions and the early events of HCV infection (reviewed in reference 2). Finally, in 2005 fully permissive cell culture systems which are based on the JFH1 clone were described (33, 66, 72). This isolate replicates with unprecedented efficiency in transfected Huh7 human hepatoma cells and produces particles infectious both in vitro and in vivo, thus providing a model system reproducing the complete HCV replication cycle.Use of these novel models has considerably expanded our knowledge of viral and host cell factors involved in HCV replication (for a recent review, see reference 59). It is now known that similar to virtually all other plus-strand RNA viruses, HCV induces intracellular membrane alterations and replicates its genome in conjunction with vesicular membrane structures, the so-called “membranous web” (10, 13). Presumably as a consequence of this specific, rather secluded architecture of the membrane-associated replication machinery, all viral proteins involved in RNA replication, with the exception of NS5A function in cis, cannot be complemented in trans (1). Restricted trans-complementation of viral replicase proteins has been observed for other plus-strand RNA viruses as well, thus indicating that a rather “closed” replication machinery is a shared feature of these viruses (15, 27, 60). In contrast, for a number of plus-strand RNA viruses from diverse virus families like Picornaviridae (poliovirus), Alphaviridae (Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus), Coronaviridae (human coronavirus E229), and Flaviviridae (tick-borne encephalitis virus, Kunjin virus, West Nile virus, and yellow fever virus), assembly of progeny viruses can be achieved when structural proteins are expressed in trans and independent from the RNA molecule that encodes the replicase proteins. Similarly, Miyanari recently reported that HCV genomes with lethal mutations in core protein can be rescued by ectopic expression of functional core protein (39). This flexibility has been extensively used to create viral vectors for gene delivery as well as viral vector-based immunization approaches (32, 48, 49, 61, 68) (for a recent review on alphaviral vectors, the most frequently used among plus strand RNA vectors, see reference 37). In these systems the viral genome region encoding the structural proteins is replaced by a transgene. The resulting defective vector genomes are capable of RNA replication but due to the lack of structural proteins are unable to produce progeny virus particles. This defect is rescued by expression of the structural proteins in trans via helper viruses (28, 55) or, in some cases, by DNA constructs stably expressed in packaging cell lines (17). The resulting virus-like particles are infectious but support only single-round infection and are unable to spread, thus improving the safety of the viral transduction system.Given the success of plus-strand RNA vector technology for basic and applied clinical research, in this study we developed a trans-complementation system for HCV that provided new insights into the basic principles of HCV particle assembly.     

Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.Hepatitis C virus (HCV) is an enveloped virus belonging to the family Flaviviridae. The virus genome is a positive-stranded RNA of about 9,600 nucleotides, which contains a single open reading frame (ORF) encoding both structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (47). Two highly conserved untranslated regions (UTRs) are found at the 5′ and 3′ ends, which play critical roles in both viral translation and replication (13, 14, 23).HCV is estimated to infect 170 million people worldwide (1, 43) and in a high percentage of individuals causes a chronic liver infection that frequently evolves into an array of diseases, including cirrhosis (12, 15, 38) and hepatocellular carcinoma (4, 7, 17, 30, 38, 49).The HCV RNA-dependent RNA polymerase (NS5B) has a high frequency of incorrect nucleotide insertions, in the range of 10⁻⁴ to 10⁻⁵ base substitutions per site, which can result in the rapid generation of HCV quasispecies (37, 45). Because of this huge genetic diversity, HCV is currently classified into six major genotypes and more than 80 subtypes (44). Recombination may be another mechanism exploited by HCV to increase genetic diversity: naturally occurring intergenotypic recombinant viruses that often have their recombination points in the trans-membrane domains of NS2 were recently identified (20, 21, 26, 27, 33, 35).Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5, 16, 36, 54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54). The relative abundance and persistence of such subgenomic RNAs in vivo suggests that (i) they are capable of autonomous replication and (ii) they can be packaged and secreted in infectious viral particles, presumably harboring the envelope proteins from wt virus coinfecting the same cell.Analysis of the genetic structure of 18 independent subgenomic deletion-containing RNAs (5, 16, 36, 54) strongly suggests that the possibility of recombination and/or deletion is restricted to specific regions.As expected, the 5′ UTR, the 3′ UTR, and the region coding from NS3 to NS5B are always conserved, in line with the notion that these regions are the minimal requirements for RNA replication. In addition to the regions required for RNA replication, however, naturally occurring subgenomic HCV RNA invariably contains an intact core region and the protease domain of NS2. In fact, the 5′ deletion boundary falls between the codons for amino acid 189 of core and amino acid 4 of E1 in 33% of cases and between the codons for amino acids 21 and 29 of E1 in 39% of cases, such that in 72% of cases overall, the 5′ boundary is between the codons for amino acid 189 of core and amino acid 29 of E1 (see Fig. S1 in the supplemental material). Likewise, the 3′ boundary shows a distinct localization, occurring between the codons for amino acids 51 and 79 of NS2 in 56% of cases.In the present study, we modified the infectious isolate JFH-1 in order to recapitulate in vitro the genetic structure of two of the most representative in-frame natural subgenomic deletion-containing RNAs found circulating in patients (28, 51, 57). Using this system, we analyzed natural subgenomic variants for their ability to replicate autonomously and demonstrated that the natural subgenomic deletion mutants are replication competent and are trans-packaged into infectious virions when coexpressed together with wt virus. Furthermore, our data suggest that the presence of the NS2 protease domain is required in order to generate the correct NS3 N terminus, required for RNA replication. Unexpectedly, the presence of NS2 generates, in turn, a strict cis requirement for the core region in order to allow efficient trans-packaging of the subgenomic RNA, revealing a complex interplay between the NS2 and the core viral genes. Finally, we performed trans-complementation studies of the subgenomic deletions with different HCV structural proteins to gain insight into the minimal requirements for the assembly and release of HCV virions.     

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.     

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5′ and the 3′ untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8, 36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replication of the viral RNA. Expression of the luciferase gene acts as a surrogate marker for levels of HCV RNA produced in the cell. The goal of the present study was to use this subgenomic HCV replicon to screen siRNA libraries and identify novel host proteins that are involved in HCV replication.A number of cellular pathways and proteins that play critical roles in HCV replication have recently been described (41, 42, 46). In particular, replication of HCV is tied closely to its localization and transport to various internal membranes and to lipid metabolism (2). Most of the HCV proteins appear to be targeted to the surface of the ER and replication complexes appear to be transported to lipid rafts, where RNA replication can occur (2). Infectious virus particle formation occurs in association with lipid droplets, and this process requires the core and NS5A proteins. In addition, cholesterol pathway production of geranylgeranyl-PP is important to geranylate the FBL2 protein, which serves as a membrane anchor for NS5A (62). The hVAP proteins involved in the localization and trafficking between internal membranous structures are known to be associated with the HCV proteins NS5A and NS5B (59). Thus, host factor lipid metabolism and intracellular protein transport are necessary for HCV replication in cells.Targeting host factors that are required for viral replication offers a strategy to overcome viral resistance and may allow treatment for more than one genotype of HCV and/or a related Flaviviridae virus such as Dengue, West Nile, or yellow fever virus. The current standard-of-care treatment for the genotype 1 strain of HCV infection is pegylated interferon alpha plus ribavirin over a 6-month time course with more than half of infected patients being refractory to this treatment (57). In addition to genotype 1, there are at least five naturally occurring genotype variants of HCV that can complicate a patient''s response to therapy when infected with more than one genotype. As well as the development of mutations, the presence of multiple variants coexisting in patients is thought to contribute to the rapid development of resistance (40). A variety of antiviral therapeutic strategies aim to inhibit viral proteins directly with small molecules or siRNAs (13, 31, 33). Although some small molecule approaches have been successful in preclinical studies, small-molecule strategies directed against the viral targets can still be rendered ineffective due to the development of mutant, treatment-resistant viral strains (13, 40). Thus, combination therapies are a necessary approach to treat the many variants of HCV that exist in the patient population.In the present study, a set of 779 SMARTpool small interfering RNAs (siRNAs) targeting the kinome and 4 siRNAs targeting 5,000 druggable genes (20,000 siRNAs) were tested for their ability to block replication of the Luc-1b HCV subgenomic replicon. siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12, 54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19). Inhibition of the cholesterol biosynthesis pathway and host cell geranylation has been previously reported to inhibit HCV subgenomic replication (3, 24, 51, 62, 67). siRNA-mediated knockdown of the class III phosphatidylinositol 4-kinases PI4KA and PI4KB inhibited luciferase expression not only for the genotype 1b subgenomic replicons (Luc-1a and Luc-1b) but also for the viral RNA levels of SG-1b, Luc-1b, and Luc-1a. PI4KA knockdown also inhibited Renilla expression in the JFH-1 genotype 2a infectious virus (JFH1-2a), genotype 2a subgenomic replicon (SG-1a), and a genomic and subgenomic genotype 1a replicon (FL-1a and SG-1a). Using the small-molecule inhibitor PIK93 in compound affinity competition experiments and in vitro biochemical assays, we demonstrated PIK93 could bind and inhibit both PI4KA and PI4KB enzymatic activity (58). PIK93 could inhibit luciferase expression in the Luc-1b, Luc-1a, and JFH1-2a infectious virus assays in the submicromolar range. Together, our data suggest that PI4KA and PI4KB regulate HCV replication and that pharmacological inhibition of these enzymes represents a new class of antiviral agents for multiple genotypes of HCV. Finally, since PI4KA and PI4KB are known to regulate protein and lipid transport to and from the ER and Golgi bodies, their function may hold clues as to how movement of HCV replication complexes throughout different organelles is regulated.