The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin beta1 and importin alpha/beta -Recognised Nuclear Localization Signals

Abstract

Dengue 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. Show more