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Journal of Virology, February 2006, p. 1121-1129, Vol. 80, No. 3
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.3.1121-1129.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
A Methionine-Rich Domain Mediates CRM1-Dependent Nuclear Export Activity of Borna Disease Virus Phosphoprotein
Hideyuki Yanai,1,
Takeshi Kobayashi,1,
,
Yohei Hayashi,1
Yohei Watanabe,1
Naohiro Ohtaki,1
Guoqi Zhang,1
Juan Carlos de la Torre,2
Kazuyoshi Ikuta,1 and
Keizo Tomonaga1*
Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, Suita, Osaka 565-0871, Japan,1 Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California2
Received 9 July 2005/ Accepted 3 November 2005
Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that replicates and transcribes in the nucleus of infected cells. Recently, we have demonstrated that BDV phosphoprotein (P) can modulate its subcellular localization through binding to the protein X, which is encoded in the overlapping open reading frame (T. Kobayashi et al., J. Virol. 77:8099-8107, 2003). This observation suggested a unique strategy of intracellular trafficking of a viral protein that is essential for the formation of a functional BDV ribonucleoprotein (RNP). However, neither the mechanism nor the consequences of the cytoplasmic retention or nuclear export of BDV X-P complex have been elucidated. In this study, we show that BDV P contains a bona fide nuclear export signal (NES) and can actively shuttle between the nucleus and cytoplasm. A transient transfection analysis of cDNA clones that mimic the BDV bicistronic X/P mRNA revealed that the methionine-rich (MetR) domain of P is responsible for the X-dependent cytoplasmic localization of the protein complex. Mutational and functional analysis revealed that the methionine residues within the MetR domain are critical for the activity of the NES of P. Furthermore, leptomycin B or small interfering RNA for inhibition of CRM1 strongly suggested that a CRM1-dependent pathway mediates nuclear export of P. Fluorescence loss in photobleaching analysis confirmed the nucleocytoplasmic shuttling of P. Moreover, we revealed that the nuclear export of P is not involved in the inhibition of the polymerase activity by X in the BDV minireplicon system. Our results may provide a unique strategy for the nucleocytoplasmic transport of viral RNP, which could be critical for the formation of not only infectious virions in the cytoplasm but also a persistent viral state in the nucleus.
* Corresponding author. Mailing address: Department of Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81 6 6879 8308. Fax: 81 6 6879 8310. E-mail: tomonaga{at}biken.osaka-u.ac.jp.
H.Y. and T. K. contributed equally to this work.
Present Address: Department of Microbiology and Immunology, Vanderbilt University Medical Center, D-7235 Medical Center North Nashville, TN 37232-2581.
Journal of Virology, February 2006, p. 1121-1129, Vol. 80, No. 3
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.3.1121-1129.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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