Brooks, Andrew JJohansson, MagnusJohn, AnnaXu, YibinJans, David AVasudevan, Subhash2015-12-130021-9258http://hdl.handle.net/1885/73072Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37-amino acid interdomain spacer sequence (residues369X2KKX14KKKX11 RKX3405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, β-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin α/β heterodimeric nuclear import receptor. We also show that the importin β-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin β, and able to target β-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin β in the infectious cycle.Keywords: Nuclear localization signals; Diseases; Enzymes; RNA; Viruses; Yeast; Physiology; beta galactosidase; helicase; hybrid protein; importin beta1; karyopherin; karyopherin alpha; protein NS5; RNA directed RNA polymerase; spacer DNA; unclassified drug; virus200210.1074/jbc.M2049772002015-12-11