Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal
Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal
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DOI：
10.1128/jvi.01850-06
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发表时间：
2007-03-01
影响因子：
5.4
通讯作者：
Wilson, Van G.
中科院分区：
文献类型：
--
作者：
Bian, Xue-Lin;Rosas-Acosta, German;Wilson, Van G.
乳头瘤病毒DNA复制发生在受感染细胞的细胞核中，需要病毒E1蛋白，该蛋白进入宿主上皮细胞的细胞核并执行启动病毒DNA复制所需的酶促功能。在本研究中，我们研究了 I 型牛乳头瘤病毒 (BPV1) 的 El 蛋白入核的途径和调控。使用体外结合测定，我们确定E1蛋白通过其核定位信号(NLS)序列与输入蛋白α3、α4和α5相互作用。与该结果一致，在与导入蛋白α3、α4或α5以及其他必要的导入因子一起温育后，纯化的E1蛋白被有效地导入洋地黄皂苷透化的HeLa细胞的细胞核中。我们还观察到，通过在 NLS 区域引入假磷酸化突变，E1 蛋白与所有三种 α 输入蛋白的体外结合显着降低。与结合缺陷一致，假磷酸化的El蛋白在体外未能进入洋地黄皂苷透化的HeLa细胞的细胞核。同样，假磷酸化突变体在体内表现出异常的细胞内定位，并且主要在转染的HeLa细胞中积聚在核膜上，而相应的丙氨酸替代突变体则表现出与野生型E1蛋白相同的细胞定位模式。总的来说，我们的数据表明 BPV1 E1 蛋白可以通过多个输入蛋白 α 转运到细胞核中，并表明宿主细胞激酶的 E1 磷酸化在调节 E1 核细胞质定位中发挥调节作用。核E1蛋白摄取的这种磷酸调节可能有助于病毒复制与角质形成细胞增殖和分化的协调。
Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral El protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the El protein from bovine papillomiavirus type I (BPV1). Using an in vitro binding assay, we determined that the El protein interacted with importins alpha 3, alpha 4, and alpha 5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified El protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin alpha 3, alpha 4, or alpha 5 and other necessary import factors. We also observed that in vitro binding of El protein to all three alpha importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated El protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type El protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin alpha and suggest that El phosphorylation by host cell kinases plays a regulatory role in modulating El nucleocytoplasmic localization. This phosphoregulation of nuclear El protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.