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

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.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…Although NS5A-PI4KIII␣ interaction occurs through NS5A domain I, it is also required for proper membranous web morphology, and therefore, we assessed the effect of I315G and P314A mutations on PI4KIII␣ activity by quantifying the intracellular PI4P levels. As expected, expression of WT NS3-5B enhanced PI4P levels (2.6-fold on average) and induced its dispersion throughout the cytoplasm with partial colocalization with NS5A (47). Mutants I315G and P314A also stimulated PI4P production (3.2-and 1.8-fold, respectively) compared with control cells, albeit to different extents (data not shown).…”

“…9A). NS5A localization pattern in cells expressing NS3-5B WT is characterized by a dispersed distribution of dot-like structures, similar to that described in replicon cells (47) (Fig. 9B, left column).…”

“…A NS5A "cluster" phenotype similar to that of the PW turn mutants was previously found in cells treated with NS5A or Cyp inhibitors (46,52,53), as well as in PI4KIII␣ knockdown cells. This phenotype was shown to correspond to lipid droplets or aberrant double membrane vesicles, respectively (47). Both CypA and PI4KIII␣ are essential host cell factors for HCV replication.…”

“…Cells were transfected with pTM-NS3-5B-based expression vectors by using the TransIT LT1 transfection reagent (Mirus Bio LLC, Madison, WI) according to the providers' instructions. Immunofluorescence staining for detection of NS5A was performed as described elsewhere (47). Briefly, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 50 g/ml digitonin for 5-10 min.…”

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…This suggests that NS5A inhibitors disturb the functional interaction of NS5A with PI4KIIIa in a similar manner to a class of experimental mutations in NS5A that also limit PI4P accumulation and inhibit HCV RNA replication and NS5A hyperphosphorylation. 18 Although inhibitor-mediated disruption of functional NS5A-PI4KIIIa interaction may contribute to the anti-HCV properties of these inhibitors, it is possible that this effect is one of several consequences of inhibitor-dependent disruption of the overall structure and/ or flexibility of NS5A. In line with this, other important interactions of NS5A with host factors (VAP-A, VAP-B, ANXA2, oxysterol binding protein, etc) may also be directly or indirectly disrupted by inhibitor binding.…”