Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication

Abstract

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha colocalized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.antivirals ͉ membranous web ͉ PI4K-IIIa ͉ PIK4CA ͉ RNAi H CV depends on cellular membrane-trafficking pathways at each step of its life cycle, including clathrin-mediated endocytosis, lipid droplet formation for virion assembly, and lipoprotein secretory pathways for egress (1-3). Like all positivestranded RNA viruses, HCV reorganizes intracellular membranes to establish sites of viral replication (4). HCV proteins are synthesized first at the rough endoplasmic reticulum (ER). Then the nonstructural proteins form viral replication complexes. HCV replication is thought to be intimately associated with modified cellular membranes that have protease-and nucleaseresistant properties (5, 6). Expression of the entire HCV polyprotein or the nonstructural protein NS4B alone results in formation of a unique multi-vesiculated structure termed the ''membranous web'' thought to be derived, at least in part, from ER membrane (6-8). Studies show that nonstructural proteins and viral RNA synthesis are associated with membranous webs (7,9). For these reasons they are implicated as the sites of active viral replication, although the precise origin and composition of the web-associated vesicles remains unclear.Cytosolic membrane rearrangements are a con...

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].…”

Hepatitis c virus and host cell lipids: An intimate connection

“…First, PI4K-IIIα colocalizes with both NS5A and double-strand (viral) RNA upon JFH-1 infection. 133 This recruitment is dependent on NS5A-D1, which is also sufficient to bind to PI4K-IIIα. 131 Second, NS5A can stimulate PI4K-IIIα activity in vitro.…”

“…132 The importance of PI4K-IIIα for HCV replication is well established, because this kinase has been identified in several siRNA-based screens as one of the top hits, regardless of the viral genotype and the experimental system. [133][134][135][136][137][138] In contrast, the role of the Golgi-resident PIK4-IIIβ is still controversially debated; it appears to contribute to replication of genotype 1 replicons, but not to replication of the JFH-1 genotype 2a isolate. 131,135,136 Nevertheless, the two isoforms of the kinase are believed to perform complementary, non-redundant tasks, because silencing of each gene decreases the levels of PI(4)P in HCV replicon cells, 81 and because the inhibitory effect mediated by PI4K-IIIα knockdown on HCV replication cannot be rescued by overexpression of PIK4-IIIβ.…”

“…131 Third, inhibition of PI4K-IIIα by RNAi alters both NS5A subcellular localization and MW architecture, 131,137 and eventually also its formation. 133 What could be the role of elevated PI(4)P levels in the HCV replication compartment? In one scenario the specific PIP composition might directly influence membrane bending and thus contribute to membrane curvature necessary for MW formation.…”

A new paradigm in viral resistance

“…Specific cellular factors used by several picornaviruses (such as PV and CVB3) and flaviviruses (such as HCV) have been identified and their mechanistic roles and potential as therapeutic targets are currently being evaluated. Of note is that a microenvironment rich in phosphatidylinositol-4-phosphate (PI4P), established through the recruitment of the phosphatidylinositol-4-kinase IIIα (PI4KIIIα) and/or IIIβ (PI4KIIIβ), is crucial for replication of several of these viruses [7][8][9][10][11][12][13]. Therefore, inhibitors targeting these components would offer broad-acting therapeutic potential.…”