title: A methionine-rich domain mediates CRM1-dependent nuclear export activity of Borna disease virus phosphoprotein.
abstract: 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.
authors.affiliation: Department of Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yanai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Yohei
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Yohei
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Naohiro
authors.initials: N
authors.lastname: Ohtaki
authors.forename: Guoqi
authors.initials: G
authors.lastname: Zhang
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: de la Torre
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ikuta
authors.forename: Keizo
authors.initials: K
authors.lastname: Tomonaga
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001891:Borna disease virus / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000821:virology; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
chemical_list: D028884:Karyopherins; D008715:Methionine; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D014764:Viral Proteins; D000097606:Exportin 1 Protein