Dataset Information

NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase III? complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

ABSTRACT: The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase III? (PI4KIII?) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIII? complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIII?-targeting inhibitor. In addition, both the NS5A and PI4KIII? classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIII?. Because of the similarities between the effects induced by treatment with PI4KIII? or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIII? complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIII? complex.

SUBMITTER: Reghellin V

PROVIDER: S-EPMC4249536 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

Reghellin V V Donnici L L Fenu S S Berno V V Calabrese V V Pagani M M Abrignani S S Peri F F De Francesco R R Neddermann P P

Antimicrobial agents and chemotherapy 20140915 12

The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hy ...[more]

PMID: 25224012

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

Dataset Information

NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase III? complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

Publications

NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

Similar Datasets

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