López Tomás, Rojas Margarito, Ayala-Bretón Camilo, López Susana, Arias Carlos F
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos 62210, Mexico.
J Gen Virol. 2005 Jun;86(Pt 6):1609-1617. doi: 10.1099/vir.0.80827-0.
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.
轮状病毒RRV基因11编码两种非结构蛋白，即NSP5和NSP6。NSP5是一种磷酸化的非结构蛋白，能以非特异性方式结合单链和双链RNA。该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（即病毒工厂）的形成提供了证据，病毒工厂被认为是病毒复制的关键结构。NSP6是一种功能未知的蛋白，似乎对细胞培养中的病毒复制并非必不可少。为了在病毒感染的背景下研究NSP5的功能，通过RNA干扰使RRV基因11的表达沉默。免疫印迹和免疫荧光分析显示，NSP5合成的减少与病毒工厂数量和大小的减少以及其他与病毒工厂相关蛋白的细胞内分布改变相关。基因11的沉默还导致病毒RNA(+)、双链RNA以及所有病毒蛋白的合成减少，同时传染性病毒的产生也减少。当兔轮状病毒阿拉巴马株的NSP5编码基因沉默时，也观察到了类似的表型。轮状病毒阿拉巴马株的NSP5基因缺乏完整NSP6蛋白的AUG起始密码子，这一事实表明，在基因11沉默的细胞中所描述的表型主要是由于缺乏NSP5所致。这项工作所呈现的数据表明，NSP5是轮状病毒复制周期中的关键蛋白。
