title: Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation.
abstract: The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6-35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany.
authors.forename: Rike
authors.initials: R
authors.lastname: Webel
authors.forename: Jens
authors.initials: J
authors.lastname: Milbradt
authors.forename: Sabrina
authors.initials: S
authors.lastname: Auerochs
authors.forename: Vera
authors.initials: V
authors.lastname: Schregel
authors.forename: Christian
authors.initials: C
authors.lastname: Held
authors.forename: Katharina
authors.initials: K
authors.lastname: Nöbauer
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Razzazi-Fazeli
authors.forename: Christophe
authors.initials: C
authors.lastname: Jardin
authors.forename: Thomas
authors.initials: T
authors.lastname: Wittenberg
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sticht
authors.forename: Manfred
authors.initials: M
authors.lastname: Marschall
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D018387:Codon, Initiator; D005347:Fibroblasts / Q000821:virology; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D017384:Sequence Deletion
chemical_list: D018387:Codon, Initiator; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase