Serotype-specific regulation of dengue virus NS5 protein subcellular localization

Dengue virus (DENV) non-structural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is known to be localized to the nucleus of infected cells in the case of DENV2, 3 and 4, whereas DENV1 NS5 is predominantly cytoplasmic. We still have an incomplete understanding of how DENV NS5 nuclear localization is regulated. Within NS5, 2 putative nuclear localization signal (NLS) sequences have been identified; NLSCentral, residing in the palm of the RdRp domain, as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single point mutations to the NLSC-term. Here, we present biochemical, virological and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the 4 DENV serotypes; DENV1 NS5 subcellular localization may occur through a weak interaction between importin-alpha (IMPα) and NLSCentral, DENV2 NS5 is almost exclusively nuclear through strong interaction between IMP and NLSC-term, while both NLSs of DENV3 NS5 contribute to directing its nuclear localization. Lastly, in the case of DENV4, NS5 nuclear localization appears to be associated with the C-terminal region and dependent on either a post-translational modification or an IMPα-independent pathway