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Journal of Virology, January 2008, p. 710-718, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.00736-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal{triangledown}

Yuji Isegawa,1* Yoichi Miyamoto,2 Yoshinari Yasuda,2 Katsunori Semi,1,5 Kenji Tsujimura,1,5 Rikiro Fukunaga,3 Atsushi Ohshima,5 Yasuhiro Horiguchi,4 Yoshihiro Yoneda,2 and Nakaba Sugimoto1

Department of Infectious Disease Control, G-5, Graduate School of Medicine,1 Biomolecular Dynamics Group,2 Laboratory of Genetics, Graduate School of Frontier Biosciences,3 Division of Toxicology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan,4 Gene Engineering Laboratory, Genomics Program, Nagahamabio Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan5

Received 5 April 2007/ Accepted 31 October 2007

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}/β in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-{alpha} subtype, NPI2/importin-{alpha}7 elicited more efficient transport activity than did Rch1/importin-{alpha}1 or Qip1/importin-{alpha}3. These results suggest a relationship between the localization of NPI2/importin-{alpha}7 and the cell tropism of HHV-6.

* Corresponding author. Mailing address: Department of Infectious Disease Control, G-5, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3301. Fax: 81-6-6879-3309. E-mail: isegawa{at}bact.med.osaka-u.ac.jp

{triangledown} Published ahead of print on 14 November 2007.

Journal of Virology, January 2008, p. 710-718, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.00736-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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