SUMMARY Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

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 ϳ9,600-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 polymer...

The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIII␣) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIII␣ is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIII␣ in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIII␣ mutants for subcellular localization, reconstitution of HCV replication in PI4KIII␣ knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIII␣ are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIII␣ core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIII␣ also were detrimental for enzymatic activity and for the ability of PI4KIII␣ to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIII␣ is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIII␣ C terminus, suggesting a catalytic center formed by an N-and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCEThe lipid kinase PI4KIII␣ is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIII␣ mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication. W orldwide, about 170 million people are chronically infected with hepatitis C virus (HCV). HCV is a positive-strand RNA virus and belongs to the family Flaviviridae. The viral genome encompasses about 9.6 kb and codes mainly for a polyprotein of about 3,000 amino acids (aa) that is flanked by nontranslated regions. The polyprotein is cleaved into 10 mature proteins by cellular and viral proteases: core, envelope glycoprotein 1 (E1) and E2, p7, and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed...

It has been demonstrated that both uncleaved, enzymitically inactive NS2/3 and cleaved NS2 proteins are rapidly degraded upon expression in cells, phenomena described to be blocked by the addition of proteasome inhibitors. As this degradation and its regulation potentially constitute an important strategy of the hepatitis C virus (HCV) to regulate the levels of its non-structural proteins, we further investigated the turnover of these proteins in relevant RNA replication systems. A lysine-mutagenesis approach was used in an effort to prevent protein degradation and determine any effect on various steps of the viral replication cycle. We show that, while NS2-lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilization of this protein, the increased NS2/3 levels do not rescue the inability of NS2/3 protease inactive replicons to replicate, suggesting that uncleaved NS2/3 is unable to functionally replace NS3 in RNA replication. Furthermore, we show that the cleaved NS2 protein is rapidly degraded in several transient and stable RNA replicon systems and that NS2 from several different genotypes also has a short half-life, highlighting the potential importance of the regulation of NS2 levels for the viral life cycle. However, in contrast to uncleaved NS2/3, neither ubiquitin nor proteasomal degradation appear to be significantly involved in NS2 degradation. Finally, although NS2 lysine-to-arginine mutagenesis does not affect this protein's levels in a JFH-1 cell culture infection system, several of these residues are identified to be involved in virion assembly, further substantiating the importance of regions of this protein for production of infectious virus.