Interaction of cellular poly(C)-binding protein 2 with nonstructural protein 1β is beneficial to Chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication
Interaction of cellular poly(C)-binding protein 2 with nonstructural protein 1β is beneficial to Chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication
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DOI：
10.1016/j.virusres.2012.08.002
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发表时间：
2012-10-01
期刊：
影响因子：
5
通讯作者：
Yang, Hanchun
中科院分区：
文献类型：
--
作者：
Wang, Lin;He, Qing;Yang, Hanchun
猪繁殖与呼吸综合征病毒（PRRSV）的非结构蛋白1 β (Nsp1 β)被认为参与抑制宿主先天免疫反应和介导病毒亚基因组mRNA转录。本研究采用酵母双杂交筛选肺泡巨噬细胞（PAMs） cDNA文库和共免疫沉淀（Co-IP）法，分析了中国高致病性PRRSV (HP-PRRSV) Nsp1 β与细胞多(C)结合2 （PCBP2）的相互作用。结果表明，HP-PRRSV的Nsp1 β在感染细胞和质粒转染细胞中都能与细胞PCBP2强烈结合并相互作用。它们的最小结合区分别为Nsp1 β的85 ~ 203aa （PCP β和CTE结构域）和PCBP2的96 ~ 168aa （KH2结构域）。接下来，我们使用共聚焦免疫荧光分析发现，在MARC-145细胞和/或质粒转染的细胞中，PRRSV感染时，Nsp1 β和PCBP2主要共定位于细胞质和核周模式。此外，sirna介导的PCBP2基因沉默在MARC-145细胞中导致病毒上清滴度和病毒蛋白显著降低，而对I型干扰素α和干扰素β的表达没有显著影响，这表明Nsp1 β与细胞PCBP2的相互作用有利于中国HP-PRRSV在MARC-145细胞中的复制。(C) 2012 Elsevier B.V.版权所有
Non-structural protein1 beta (Nsp1 beta) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be involved in suppressing the host innate immune response and mediating viral subgenomic mRNA transcription. In the present study, we have analyzed the interaction of Nsp1 beta of Chinese highly pathogenic PRRSV (HP-PRRSV) with cellular poly(C)-binding 2 (PCBP2) by means of the yeast two-hybrid screening in a pulmonary alveolar macrophages (PAMs) cDNA library and co-immunoprecipitation (Co-IP) assay. Our results indicated that the Nsp1 beta of the HP-PRRSV is able to bind and interact with cellular PCBP2 strongly in both the infected cells and plasmid transfected cells. Their minimal binding regions were identified to be the residues 85-203 aa (PCP beta and CTE domains) for the Nsp1 beta and the residues 96-168 aa (KH2 domain) for PCBP2, respectively. Next, we used confocal immunofluorescence analysis and discovered that, during PRRSV infection in MARC-145 cells and/or pasmid-transfected cells, the Nsp1 beta and PCBP2 mainly colocalized in the cytoplasm and perinuclear pattern. Moreover, the siRNA-mediated silencing of PCBP2 gene in the MARC-145 cells resulted in significant reduction of the virus titer in supernatants as well as viral proteins, while no significant effects on the expression of the type I interferon alpha and interferon beta, suggesting that the interaction of the Nsp1 beta with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells. (C) 2012 Elsevier B.V. All rights reserved.