Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication.
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.
Berger
Kristi L
KL
Department of Microbiology, University of Chicago, Chicago, IL 60637, USA.
Cooper
Jacob D
JD
Heaton
Nicholas S
NS
Yoon
Rosa
R
Oakland
Todd E
TE
Jordan
Tristan X
TX
Mateu
Guaniri
G
Grakoui
Arash
A
Randall
Glenn
G
D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D004705:Endocytosis; D011992:Endosomes / Q000821:virology; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D012324:RNA-Dependent RNA Polymerase / Q000502:physiology; D014779:Virus Replication
D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D012324:RNA-Dependent RNA Polymerase