title: Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal.
abstract: To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.
authors.affiliation: Department of Infectious Disease Control, G-5, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. isegawa@bact.med.osaka-u.ac.jp
authors.forename: Yuji
authors.initials: Y
authors.lastname: Isegawa
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamoto
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Yasuda
authors.forename: Katsunori
authors.initials: K
authors.lastname: Semi
authors.forename: Kenji
authors.initials: K
authors.lastname: Tsujimura
authors.forename: Rikiro
authors.initials: R
authors.lastname: Fukunaga
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ohshima
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Horiguchi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
authors.forename: Nakaba
authors.initials: N
authors.lastname: Sugimoto
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C464753:pU69 kinase, human herpesvirus 6