Dengue virus nonstructural
protein 5 (NS5) is a large multifunctional
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 beta1 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
green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length
complementary DNA. 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 (K(387)K(388)K(389)); 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 with wild-type virus expressing nuclear-localizing NS5, to reduce
interleukin-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.