Gene-specific inhibition of reovirus replication by RNA interference
Gene-specific inhibition of reovirus replication by RNA interference
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DOI：
10.1128/jvi.00276-06
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发表时间：
2006-09-01
影响因子：
5.4
通讯作者：
Dermody, Terence S.
中科院分区：
文献类型：
--
作者：
Kobayashi, Takeshi;Chappell, James D.;Dermody, Terence S.
哺乳动物呼肠孤病毒含有10段双链RNA （dsRNA）的基因组。呼肠孤病毒的复制和组装发生在被感染细胞的细胞质中形成的称为病毒包涵体的独特结构中。病毒非结构蛋白mu NS和sigma NS以及核心蛋白mu 2在形成病毒包涵体和招募其他病毒蛋白和RNA到这些结构中进行复制和组装方面发挥关键作用。然而，这些蛋白质在病毒复制中的确切功能还不清楚。因此，为了更好地了解呼肠病毒蛋白与病毒包涵体形成相关的功能，我们使用基于质粒的载体建立了稳定表达小干扰rna （sirna）的293T细胞系，sirna特异性编码菌株3型Dearing （T3D）的mu 2、mu NS和sigma NS蛋白的转录本。传染性实验显示，在稳定表达mu 2、mu NS或sigma NS siRNA的293T细胞中，T3D的产量显著降低，而菌株1 Lang的产量不显著降低。这些蛋白特异性sirna的稳定表达大大减少了病毒的dsRNA、蛋白质合成和包涵体形成，表明它们都是病毒复制机制的关键组成部分。利用稳定表达mu NS siRNA的细胞系，我们开发了一种互补系统，通过瞬时转染重组T3D mu NS，在RNS siRNA靶向的序列中引入沉默突变，来挽救病毒复制。此外，我们证明了mu NSC缺乏mu NS的前40个氨基残基，不能恢复呼肠孤病毒在互补系统中的生长。这些结果揭示了病毒包涵蛋白相互依赖的功能，并表明稳定表达呼肠孤病毒sirna的细胞系是研究病毒蛋白结构-功能关系的有用工具。
Mammalian reoviruses contain a genome of 10 segments of double-stranded RNA (dsRNA). Reovirus replication and assembly occur within distinct structures called viral inclusions, which form in the cytoplasm of infected cells. Viral nonstructural proteins mu NS and sigma NS and core protein mu 2 play key roles in forming viral inclusions and recruiting other viral proteins and RNA to these structures for replication and assembly. However, the precise functions of these proteins in viral replication are poorly defined. Therefore, to better understand the functions of reovirus proteins associated with formation of viral inclusions, we used plasmid-based vectors to establish 293T cell lines stably expressing small interfering RNAs (siRNAs) specific for transcripts encoding the mu 2, mu NS, and sigma NS proteins of strain type 3 Dearing (T3D). Infectivity assays revealed that yields of T3D, but not those of strain type 1 Lang, were significantly decreased in 293T cells stably expressing mu 2, mu NS, or sigma NS siRNA. Stable expression of siRNAs specific for any one of these proteins substantially diminished viral dsRNA, protein synthesis, and inclusion formation, indicating that each is a critical component of the viral replication machinery. Using cell lines stably expressing mu NS siRNA, we developed a complementation system to rescue viral replication by transient transfection with recombinant T3D mu NS in which silent mutations were introduced into the sequence targeted by the RNS siRNA. Furthermore, we demonstrated that mu NSC, which lacks the first 40 amino residues of mu NS, is incapable of restoring reovirus growth in the complementation system. These results reveal interdependent functions for viral inclusion proteins and indicate that cell lines stably expressing reovirus siRNAs are useful tools for the study of viral protein structure-function relationships.