Reduced expression of the rotavirus NSP5 gene has a pleiotropic effect on virus replication
Reduced expression of the rotavirus NSP5 gene has a pleiotropic effect on virus replication
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DOI：
10.1099/vir.0.80827-0
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发表时间：
2005-06-01
影响因子：
3.8
通讯作者：
Arias, CF
中科院分区：
文献类型：
--
作者：
López, T;Rojas, M;Arias, CF
轮状病毒RRV基因11编码NSP5和NSP6两种非结构蛋白。NSP5是一种磷酸化的非结构蛋白，以非特异性的方式与单链和双链RNA结合。这种蛋白在未感染细胞中的瞬时表达为其参与电子致密细胞质结构的形成提供了证据，这种结构被称为病毒质，被认为是病毒复制的关键结构。NSP6是一种功能未知的蛋白质，似乎对细胞培养中的病毒复制不是必需的。为了研究NSP5在病毒感染背景下的功能，通过RNA干扰沉默了RRV基因11的表达。免疫印迹和免疫荧光分析表明，NSP5合成的减少与病毒质的数量和大小减少以及其他病毒质相关蛋白在细胞内的分布改变有关。基因11的沉默还导致病毒RNA(+)和双链RNA以及所有病毒蛋白的合成减少，以及传染性病毒的产生减少。当阿拉巴马州Lapine轮状病毒株的NSP5编码基因被沉默时，也观察到了类似的表型。轮状病毒阿拉巴马州的NSP5基因缺乏完整NSP6蛋白的AUG启动子密码子，这表明在基因11沉默的细胞中所描述的表型主要是由于NSP5的缺失。这项工作提供的数据表明，NSP5是轮状病毒复制周期中的关键蛋白。
Rotavirus RRV gene 11 encodes two non-structural proteins, NSP5 and NSP6. NSP5 is a phosphorylated non-structural protein that binds single- and double-stranded RNA in a non-specific manner. Transient expression of this protein in uninfected cells has provided evidence for its participation in the formation of electron-dense cytoplasmic structures, known as viroplasms, which are thought to be key structures for the replication of the virus. NSP6 is a protein of unknown function that seems not to be essential for virus replication in cell culture. To study the function of NSP5 in the context of a viral infection, the expression of RRV gene 11 was silenced by RNA interference. Reduction in the synthesis of NSP5, as shown by immunoblot and immunofluorescence assays, correlated with a reduction in the number and size of viroplasms and with an altered intracellular distribution of other viroplasm-associated proteins. Silencing of gene 11 also resulted in a reduced synthesis of viral RNA(+) and double-stranded RNA and of all viral proteins, as well as in a decreased production of infectious virus. A similar phenotype was observed when the NSP5 coding gene of the lapine rotavirus strain Alabama was silenced. The fact that the NSP5 gene of rotavirus Alabama lacks the AUG initiator codon for a complete NSP6 protein, suggests that the described phenotype in gene 11-silenced cells is mostly due to the absence of NSP5. The data presented in this work suggest that NSP5 is a key protein during the replication cycle of rotaviruses.