The interdomain region of Dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha /beta -recognised nuclear localisation signals (NLSs)

doi:10.1074/jbc.M204977200

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The interdomain region of Dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin $beta$ 1 and importin $alpha$ / $beta$ -recognised nuclear localisation signals (NLSs)

Andrew J. Brooks, Magnus Johansson, Anna V. John, Yibin Xu, David A. Jans, and Subhash G. Vasudevan

Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811

Corresponding Author: subhash.vasudevan{at}jcu.edu.au

Dengue virus NS5 protein is a multifunctional RNA dependent RNA polymerase that is essential for virus replication. We have previously shown that the 37 amino acid interdomain spacer sequence (residues 369-405: X2KKX14KKKX11RKX3) of Dengue2 NS5 contains a functional nuclear localisation signal (NLS). In this study, $beta$ -galactosidase ( $beta$ -gal) fusion proteins carrying point mutations of the positively charged residues (bold) or truncations of the interdomain linker region (residues 369 to 389 or residues 386 to 405) were analysed for nuclear import and importin binding activities to show that the amino terminal part of the linker region (residues 369-389 – a/bNLS) is critical for nuclear localisation and is recognised with high affinity by the conventional NLS-binding importin $alpha$ / $beta$ heterodimeric nuclear import receptor. We also show that the importin $beta$ binding site (residues 320-368 – bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognised with high affinity by importin $beta$ , and able to target $beta$ -gal to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin $beta$ in the infectious cycle.

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The interdomain region of Dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin 1 and importin /-recognised nuclear localisation signals (NLSs)

The interdomain region of Dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin $beta$ 1 and importin $alpha$ / $beta$ -recognised nuclear localisation signals (NLSs)