Abstract
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.
Highlights
Worldwide about 170 million people are chronically infected with hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, frequently leading to severe liver disease
Hepatitis C virus (HCV) infections affect about 170 million people worldwide and often result in severe chronic liver disease
We and others identified the lipid kinase phosphatidylinositol-4 kinase III alpha (PI4KIIIa), catalyzing the synthesis of phosphatidylinositol 4-phosphate (PI4P), as an essential host factor involved in the formation of the membranous web
Summary
Worldwide about 170 million people are chronically infected with hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, frequently leading to severe liver disease. The structural proteins core, E1 and E2, which are the major constituents of the viral particle, p7, a presumed viroporin, and NS2, which is part of the protease mediating NS2/NS3 cleavage, are mainly involved in the generation of infectious virions, whereas NS3 to NS5B are required for RNA replication. Terminal protease, which is constitutively bound to its cofactor NS4A. NS4B plays a major role in inducing membrane alterations that are required for viral replication (reviewed in [3]). NS5A is a phosphoprotein consisting of three subdomains with functions in viral RNA replication and virus assembly (reviewed in [4]) and NS5B is the viral RNA-dependent RNA-polymerase (RdRP)
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