Global COE Program, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
J Virol. 2012 Oct;86(20):11128-37. doi: 10.1128/JVI.01700-12. Epub 2012 Aug 1.
Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is an enveloped virus containing a single-stranded, positive-sense RNA genome. Nine mRNAs carrying a set of common 5' and 3' untranslated regions (UTR) are synthesized from the incoming viral genomic RNA in cells infected with SCoV. A nonstructural SCoV nsp1 protein causes a severe translational shutoff by binding to the 40S ribosomal subunits. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNA. However, the mechanism by which SCoV viral proteins are efficiently produced in infected cells in which host protein synthesis is impaired by nsp1 is unknown. In this study, we investigated the role of the viral UTRs in evasion of the nsp1-mediated shutoff. Luciferase activities were significantly suppressed in cells expressing nsp1 together with the mRNA carrying a luciferase gene, while nsp1 failed to suppress luciferase activities of the mRNA flanked by the 5'UTR of SCoV. An RNA-protein binding assay and RNA decay assay revealed that nsp1 bound to stem-loop 1 (SL1) in the 5'UTR of SCoV RNA and that the specific interaction with nsp1 stabilized the mRNA carrying SL1. Furthermore, experiments using an SCoV replicon system showed that the specific interaction enhanced the SCoV replication. The specific interaction of nsp1 with SL1 is an important strategy to facilitate efficient viral gene expression in infected cells, in which nsp1 suppresses host gene expression. Our data indicate a novel mechanism of viral gene expression control by nsp1 and give new insight into understanding the pathogenesis of SARS.
严重急性呼吸综合征冠状病毒（SARS-CoV）是一种包膜病毒，含有一条单链、正链 RNA 基因组。在感染 SARS-CoV 的细胞中，从进入的病毒基因组 RNA 合成九条携带一组共同 5'和 3'非翻译区（UTR）的 mRNA。非结构 SARS-CoV nsp1 蛋白通过与 40S 核糖体亚基结合导致严重的翻译关闭。nsp1-40S 核糖体复合物进一步诱导宿主 mRNA 5'UTR 附近的内切核酸酶切割。然而，在 nsp1 损害宿主蛋白合成的感染细胞中，SARS-CoV 病毒蛋白如何高效产生的机制尚不清楚。在这项研究中，我们研究了病毒 UTR 在逃避 nsp1 介导的关闭中的作用。与携带荧光素酶基因的 mRNA 一起表达 nsp1 的细胞中的荧光素酶活性显着受到抑制，而 nsp1 未能抑制由 SARS-CoV 5'UTR 侧翼的 mRNA 的荧光素酶活性。RNA-蛋白结合测定和 RNA 衰变测定表明，nsp1 与 SARS-CoV RNA 的 5'UTR 中的茎环 1（SL1）结合，并且与 nsp1 的特异性相互作用稳定了携带 SL1 的 mRNA。此外，使用 SARS-CoV 复制子系统的实验表明，特异性相互作用增强了 SARS-CoV 的复制。nsp1 与 SL1 的特异性相互作用是在抑制宿主基因表达的感染细胞中促进病毒基因表达的有效策略。我们的数据表明 nsp1 对病毒基因表达的控制存在一种新机制，并为理解 SARS 的发病机制提供了新的见解。
