The Role of the Phosphatidylinositol 4-Kinase PI4KA in Hepatitis C Virus-Induced Host Membrane Rearrangement

Abstract

BackgroundHepatitis C virus (HCV), like other positive-sense RNA viruses, replicates on an altered host membrane compartment that has been called the “membranous web.” The mechanisms by which the membranous web are formed from cellular membranes are poorly understood. Several recent RNA interference screens have demonstrated a critical role for the host phosphatidylinositol 4-kinase PI4KA in HCV replication. We have sought to define the function of PI4KA in viral replication.Methodology/Principal FindingsUsing a nonreplicative model of membranous web formation, we show that PI4KA silencing leads to aberrant web morphology. Furthermore, we find that PI4KA and its product, phosphatidylinositol 4-phosphate, are enriched on membranous webs and that PI4KA is found in association with NS5A in HCV-infected cells. While the related lipid kinase PI4KB also appears to support HCV replication, it does not interact with NS5A. Silencing of PI4KB does not overtly impair membranous web morphology or phosphatidylinositol 4-phosphate enrichment at webs, suggesting that it acts at a different point in viral replication. Finally, we demonstrate that the aberrant webs induced by PI4KA silencing require the activity of the viral NS3-4A serine protease but not integrity of the host secretory pathway.Conclusions/SignificancePI4KA is necessary for the local enrichment of PI 4-phosphate at the HCV membranous web and for the generation of morphologically normal webs. We also show that nonreplicative systems of web formation can be used to order molecular events that drive web assembly.

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

“…These studies offered new possibilities to counter the establishment of viral replication machinery using PI4K inhibitors. The important feature of these studies was that viral replication was almost completely abolished by even partial PI4K knockdown, which elicited no apparent cellular toxicity in vitro (21). These findings have confirmed our previous conclusions that even small amounts of the enzyme can fulfill its "housekeeping" functions (9).…”

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…Similar to enteroviruses, the flavivirus Hepatitis C virus (HCV) requires PI4P lipids for replication, which are generated via the recruitment of PI4KIIIα [46][47][48][49][50][51][52][53]. West Nile virus attracts the cholesterol-synthesizing enzyme 3-HMGA-CoA reductase to replication sites [54,55], while dengue virus recruits fatty acid synthase and stimulates its activity [56,57].…”

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

“…siRNA screening for host factors has revealed that PI4Ks are essential for HCV proliferation (28). Because the ER-localized PI4 kinase PI4KIII␣ has been shown to be important for viral induced membrane rearrangement and HCV replication, we primarily focused on investigating the role of Golgi-localized PI4P pools (30,31). We took advantage of Golgi-specific hSac1 K2A phosphatase to specifically deplete PI4P from the TGN.…”

“…Two of these kinases, PI4KII␣ and PI4KIII␤, localize to the Golgi complex; however, PI4KIII␤ plays a more dominant role in maintaining important functional and structural attributes of the Golgi complex (29). It was recently shown that HCV induces enrichment of PI4P pools near the ER by PI4KIII␣ to trigger host membrane rearrangement required for HCV replication (30,31).…”

“…1A; see TGN46ϩPI4P panel). The ER-localized pools of PI4P have been shown to induce membrane alterations and promote HCV replication (30,31). Thus, we sought to investigate the potential role of Golgi-localized PI4P in the HCV secretion process by employing the hSac1 K2A expression strategy without affecting HCV replication.…”