Dengue virus non-structural protein NS5 is a large multi-functional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ("aNLS" and "bNLS"), that are recognized by the importin alpha/beta and importin beta nuclear transporters respectively. Here we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time, and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding GFP fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length cDNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K387 K388 K389); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear localizing NS5, as compared to wild type virus expressing nuclear localizing NS5, to reduce interleukin-8 (IL-8) production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.
|Translated title of the contribution
|Nuclear Localization of Dengue Virus Nonstructural Protein 5 Through Its Importin α/β-Recognized Nuclear Localization Sequences is Integral to Viral Infection
|795 - 807
|Number of pages
|Published - Jul 2007