title: The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized 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 (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-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 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, recognized with high affinity by importin beta, and able to target beta-galactosidase 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, James Cook University, Queensland 4811, Australia.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Brooks
authors.forename: Magnus
authors.initials: M
authors.lastname: Johansson
authors.forename: Anna V
authors.initials: AV
authors.lastname: John
authors.forename: Yibin
authors.initials: Y
authors.lastname: Xu
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003716:Dengue Virus / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D013997:Time Factors; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D005456:Fluorescent Dyes; D005982:Glutathione Transferase; D019913:Nuclear Localization Signals; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D001616:beta-Galactosidase; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C096041:NS5 protein, flavivirus