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

Published

Journal Article

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 co-localized 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.

Full Text

Duke Authors

Cited Authors

  • Berger, KL; Cooper, JD; Heaton, NS; Yoon, R; Oakland, TE; Jordan, TX; Mateu, G; Grakoui, A; Randall, G

Published Date

  • May 5, 2009

Published In

Volume / Issue

  • 106 / 18

Start / End Page

  • 7577 - 7582

PubMed ID

  • 19376974

Pubmed Central ID

  • 19376974

Electronic International Standard Serial Number (EISSN)

  • 1091-6490

Digital Object Identifier (DOI)

  • 10.1073/pnas.0902693106

Language

  • eng

Conference Location

  • United States