The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIII alpha) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIII alpha 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 PI4KIII alpha and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIII alpha interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIII alpha functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIII alpha binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIII alpha. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIII alpha affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIII alpha or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIII alpha increased relative abundance of basally phosphorylated NS5A (p56). PI4KIII alpha 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 PI4KIII alpha in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIII alpha activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIII alpha in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.