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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.