West Nile Virus (WNV) is a single stranded, positive sense RNA virus of the family Flaviviridae. The WNV genome consists of 10 genes that encode for 3 structural proteins, required for the formation of the virus, and 7 non structural proteins, that are responsible for viral replication and host modulation. It is well understood that Positive sense RNA viruses remodel intracellular membranes in the cytoplasm to induce the formation of membranous structures that constitute the replication complex. These structures are crucial for efficient viral RNA transcription, protein translation and evading immune detection. Interestingly though, some viral proteins not only localise to these membrane compartments in the cytoplasm, but are also observed to localise to the nucleus; typically having roles in attenuating the host immune response. Previously we published that the WNV NS5 did not appear within the nucleus, however upon inhibition of nuclear export with leptomycin B we observed a significant accumulation of NS5 within the nucleus. In addition, the inhibition of nuclear import lead to a severe attenuation of WNV replication in treated cells, but interestingly inhibition of nuclear export had little effect on virus replication. Gene mining revealed a putative bi-partite nuclear localisation sequence within WNV NS5, which we functionally interrogated via site-directed mutagenesis. To this end we observed that mutation of this motif and subsequent transient expression of NS5 resulted solely in cytoplasmic accumulation of NS5. supporting that this region is a functional nuclear localisation sequence. Initial introduction of these mutations into FLSDX, an infectious clone of WNV, appears lethal to replication suggesting that nuclear localisation of WNV NS5 is crucial for replication. Additionally we have shown that nuclear localization of NS5 is mediated via interaction with importin proteins. From this, we hypothesise that the nuclear localisation of WNV NS5 may have an integral role for viral replication. Thus, our aims are to further define the role NS5 has within the nucleus, and the impact of NS5 on host gene regulation and expression.