Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein

ABSTRACT Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the orderMononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.

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A Methionine-Rich Domain Mediates CRM1-Dependent Nuclear Export Activity of Borna Disease Virus Phosphoprotein

Journal of Virology ◽

10.1128/jvi.80.3.1121-1129.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1121-1129 ◽

Cited By ~ 19

Author(s):

Hideyuki Yanai ◽

Takeshi Kobayashi ◽

Yohei Hayashi ◽

Yohei Watanabe ◽

Naohiro Ohtaki ◽

...

Keyword(s):

Disease Virus ◽

Nuclear Export ◽

Rna Virus ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Polymerase Activity ◽

Cdna Clones ◽

Borna Disease Virus ◽

Rich Domain ◽

Borna Disease

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.

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Detailed Mapping of the Nuclear Export Signal in the Rous Sarcoma Virus Gag Protein

Journal of Virology ◽

10.1128/jvi.79.14.8732-8741.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 8732-8741 ◽

Cited By ~ 32

Author(s):

Lisa Z. Scheifele ◽

Eileen P. Ryan ◽

Leslie J. Parent

Keyword(s):

Rous Sarcoma Virus ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Gag Protein ◽

Hydrophobic Residues ◽

Gag Polyprotein ◽

Export Activity ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Export Signal

ABSTRACT The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.

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