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.

Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.

The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.The hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (47). The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame (ORF), flanked by nontranslated regions (NTRs), that is translated primarily into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membrane-associated replication complex, in which NS5B is the RNA-dependent RNA polymerase (RdRp), the key enzyme of viral RNA replication. Purified NS5B can initiate RNA synthesis in vitro by a primer-dependent mechanism or de novo (7,28,30,55). De novo initiation at the 3Ј end of the viral positiveand negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells. The crystal structures of several viral RdRps that initiate RNA synthesis de novo have been reported, including that of HCV NS5B (3,13,25), the first such structure to be solved, and more recently those of other Flaviviridae polymerases (15, 31, 51). All of these enzymes are homologous and for all of them the "fingers" and "thumb" subdomains are connected (through the so-called "fingertips") and cluster around the central, catalytic "palm" s...

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...

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