Ni Bing, Shi Xinfu, Li Yang, Gao Wenda, Wang Xiliang, Wu Yuzhang
Institute of Immunology PLA, Third Military Medical University, Chongqing, China.
Antivir Ther. 2005;10(4):527-33.
Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.
严重急性呼吸综合征（SARS）是一种由新型冠状病毒（SARS-CoV）引起的新发传染病，于2003年初传播到30多个国家。直到最近，尚无特异性疫苗和有效药物可保护患者免受该病毒感染。为探索对抗这种疾病的新策略，我们用噻唑蓝（MTT）、空斑形成、蛋白质免疫印迹和实时聚合酶链反应（PCR）检测法，研究了干扰RNA（RNAi）对病毒感染和复制的影响。结果显示，靶向SARS-CoV基因组非结构蛋白1（NSP1）序列的质粒来源的小干扰RNA（siRNA）可特异性抑制NSP1序列的表达，并有效抑制SARS-CoV在培养的非洲绿猴肾细胞（Vero E6）系中的复制和增殖。在用PCR和蛋白质免疫印迹检测法分析时，SARS-CoV感染3天后，表达siRNA的细胞中SARS-CoV刺突蛋白和核蛋白基因在mRNA和蛋白质水平的表达明显低于对照组。我们的研究提供了强有力的证据，证明SARS-CoV基因组中的NSP1序列是RNAi的有效靶点，且siRNA的作用可能主要源于对SARS-CoV亚基因组合成整体减少及随后蛋白质表达的影响。
