Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization

Dengue virus (DENV) nonstructural 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 localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLS_Central residing in the palm of the RdRp domain as well as the recently discovered NLS_C-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 NLS_C-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 four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLS_Central and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLS_C-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLS_C-term.