Nucleocytoplasmic shuttling of the West Nile virus RNA‐dependent RNA polymerase NS5 is critical to infection

West Nile virus (WNV) is a single‐stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNV_KUN) RNA‐dependent RNA polymerase (protein NS5) is central to WNV_KUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNV_KUN‐infected cells and NS5‐transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site‐directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNV_KUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNV_KUN, resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNV_KUN replication. This study thus shows for the first time that NLS‐dependent trafficking into the nucleus during infection of WNV_KUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNV_KUN virus production, proving the principle that inhibition of WNV_KUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.