West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis.
Using West Nile virus strain Kunjin virus (WNV(KUN)) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNV(KUN) or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [(32)P]orthophosphate revealed that WNV(KUN) NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.
Mackenzie
Jason M
JM
School of Molecular and Microbial Sciences, University of Queensland, St Lucia, QLD 4072, Australia.
Kenney
Mark T
MT
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, and Clinical Medical Virology Centre, University of Queensland, Herston, Brisbane, QLD 4029, Australia.
Westaway
Edwin G
EG
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, and Clinical Medical Virology Centre, University of Queensland, Herston, Brisbane, QLD 4029, Australia.
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002467:Cell Nucleus / Q000737:chemistry; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014902:West Nile virus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
C096041:NS5 protein, flavivirus; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase