A methionine-rich domain mediates CRM1-dependent nuclear export activity of Borna disease virus phosphoprotein
A methionine-rich domain mediates CRM1-dependent nuclear export activity of Borna disease virus phosphoprotein
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DOI：
10.1128/jvi.80.3.1121-1129.2006
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发表时间：
2006-02-01
影响因子：
5.4
通讯作者：
Tomonaga, K
中科院分区：
文献类型：
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作者：
Yanai, H;Kobayashi, T;Tomonaga, K
博尔纳病病毒(BDV)是一种在感染细胞的细胞核内复制和转录的非分段负链RNA病毒。最近，我们已经证明BDV磷酸蛋白(P)可以通过与编码在重叠开放阅读框架中的蛋白X结合来调节其亚细胞定位(T.Kobayashi等人，J.Virol)。77：8099-8107,2003)。这一观察表明了一种独特的病毒蛋白在细胞内运输的策略，这种病毒蛋白是形成功能性BDV核糖核蛋白(RNP)所必需的。然而，BDV X-P复合体胞质滞留或核输出的机制和后果尚未阐明。在这项研究中，我们发现BDV P含有一个真实的核输出信号(NES)，并能在细胞核和细胞质之间主动穿梭。对模拟BDV双顺反子X/P mRNA的cDNA克隆的瞬时转染分析表明，P的蛋氨酸富含结构域(Metr)负责该蛋白复合体的X依赖细胞质定位。突变和功能分析表明，MetR结构域中的蛋氨酸残基对P的NES活性至关重要。此外，抑制CRM1的轻霉素B或小干扰RNA强烈表明，依赖CRM1的途径介导了P的核输出。光漂白中的荧光损失证实了P的核质穿梭。此外，我们还揭示了P的核输出不参与X对BDV微型复制子系统中聚合酶活性的抑制。我们的结果可能为病毒RNP的核质运输提供了一种独特的策略，这可能不仅对细胞质中感染性病毒粒子的形成至关重要，而且对细胞核中持续的病毒状态也是至关重要的。
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.