title: Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding.
abstract: The multifunctional protein kinase pUL97 of human cytomegalovirus (HCMV) strongly determines the efficiency of virus replication. Previously, the existence of two pUL97 isoforms that arise from alternative translational initiation and show a predominant nuclear localization was described. Two bipartite nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of the large isoform, whilst the small isoform exclusively contained NLS2. The current study found the following: (i) pUL97 nuclear localization in HCMV-infected primary fibroblasts showed accumulations in virus replication centres and other nuclear sections; (ii) in a quantitative evaluation system for NLS activity, the large isoform showed higher efficiency of nuclear translocation than the small isoform; (iii) NLS1 was mapped to aa 6-35 and NLS2 to aa 190-213; (iv) using surface plasmon resonance spectroscopy, the binding of both NLS1 and NLS2 to human importin-α was demonstrated, stressing the importance of individual arginine residues in the bipartite consensus motifs; (v) nuclear magnetic resonance spectroscopy of pUL97 peptides confirmed an earlier statement about the functional requirement of NLS1 embedding into an intact α-helical structure; and (vi) a bioinformatics investigation of the solvent-accessible surface suggested a high accessibility of NLS1 and an isoform-specific, variable accessibility of NLS2 for interaction with importin-α. Thus, the nucleocytoplasmic transport mechanism of the isoforms appeared to be differentially regulated, and this may have consequences for isoform-dependent functions of pUL97 during virus replication.
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, 91054 Erlangen, Germany.
authors.forename: Rike
authors.initials: R
authors.lastname: Webel
authors.affiliation: Centre of Pharmacy and Department of Chemistry, University of Bergen, Bergen, Norway.
authors.forename: Sara M Ø
authors.initials: SMØ
authors.lastname: Solbak
authors.affiliation: Department for Image Processing and Biomedical Engineering, Fraunhofer Institute for Integrated Circuits IIS, Erlangen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Held
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, 91054 Erlangen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Milbradt
authors.affiliation: Department Medicinal Chemistry, University of Erlangen-Nuremberg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Groß
authors.affiliation: Department Medicinal Chemistry, University of Erlangen-Nuremberg, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Eichler
authors.affiliation: Department for Image Processing and Biomedical Engineering, Fraunhofer Institute for Integrated Circuits IIS, Erlangen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wittenberg
authors.affiliation: Division of Bioinformatics, Institute of Biochemistry, University of Erlangen-Nuremberg, Germany.
authors.forename: Christophe
authors.initials: C
authors.lastname: Jardin
authors.affiliation: Division of Bioinformatics, Institute of Biochemistry, University of Erlangen-Nuremberg, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sticht
authors.affiliation: Centre of Pharmacy and Department of Chemistry, University of Bergen, Bergen, Norway.
authors.forename: Torgils
authors.initials: T
authors.lastname: Fossen
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, 91054 Erlangen, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Marschall
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003198:Computer Simulation; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D019913:Nuclear Localization Signals; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D028901:alpha Karyopherins; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase