Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 18, Pages 7577-7582Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0902693106
Keywords
antivirals; membranous web; PI4K-IIIa; PIK4CA; RNAi
Categories
Funding
- The University of Chicago Cancer Research Center
- Digestive Disease Research Core Center [P30 DK42086]
- Susan and David Sherman
- Schweppe Foundation
- American Liver Foundation
- National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases Region V Great Lakes Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Research [1-U54-AI-057153]
- American Society for Microbiology's Microbiology Undergraduate Research Fellowship
- NIH Training Grant [T32 AI065382-01, T32 GM007183]
- Cancer Research Institute Investigator Award
- Public Health Service [AI070101]
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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.
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