Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.
Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.
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Importin α 5 负向调节 importin β 1 介导的新城疫病毒基质蛋白核输入以及鸡成纤维细胞中的病毒复制和致病性
DOI：
10.1080/21505594.2018.1449507
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发表时间：
2018-12-31
期刊：
影响因子：
5.2
通讯作者：
Liu X
中科院分区：
文献类型：
--
作者：
Duan Z;Xu H;Ji X;Zhao J;Xu H;Hu Y;Deng S;Hu S;Liu X
纽卡斯尔病毒（NDV）的基质（M）蛋白通过内源性核定位信号（NLS）定位于细胞核内，但参与NDV M蛋白核输入的细胞蛋白以及M蛋白核定位在NDV复制和致病中的作用尚不清楚。本研究通过酵母双杂交技术筛选出与新城疫病毒M蛋白相互作用的importin β1。这种相互作用随后通过免疫共沉淀和下拉测定法证实。体外结合研究表明，M蛋白的NLS区和属于RanGTP结合区的importin β1的336-433位氨基酸对结合有重要作用。重要的是，具有M/NLS突变的重组病毒导致NDV的致病型改变，并且减弱了NDV在鸡成纤维细胞和SPF鸡中的复制和致病性。与结合数据一致，NDV M蛋白在毛地黄皂苷透化的HeLa细胞中的核输入需要importin β1和RanGTP。有趣的是，importin α5被证实通过结合importin β1与M蛋白相互作用。而导入蛋白β1和α5的siRNA去除后，M蛋白在鸡成纤维细胞中的聚集明显，NDV的复制能力明显降低，致病性明显增强。本研究首次揭示了NDV M蛋白的核输入机制，以及importin α5在importin β1介导的M蛋白核输入和副粘病毒复制及致病性中的负调控作用。
The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.
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