质粒介导的RNAi抑制SARS-CoV复制和感染

Inhibition of replication and infection of the severe acute respiratory syndrome-associated coronavirus with plasmid-mediated RNAi

目的研究RNAi作为治疗SARS-CoV感染新策略的可能性。方法选择并克隆针对SARS-CoV编码基因的共同前导序列非结构蛋白-1(Non-structureprotein1,NSP1)的siRNAs;RT-PCR和FACS法检测所选取的siRNAs对NSP1基因抑制的特异性和有效性;利用MTT法检测选取的siRNAs对病毒感染的细胞生存率的影响;空斑形成实验证实在siRNAs存在下病毒量减少情况;RT-PCR和Western-blot法进一步证实感染细胞中S和N基因在mRNA和蛋白水平的表达情况。结果结果显示重组质粒衍生的靶向SARS-CoVNSP1的siRNAs能特异地抑制靶序列表达;在体外培养的VeroE6细胞中能显著抑制SARS-CoV的复制与繁殖。感染1d后,RT-PCR和Western-blot结果显示,同对照组相比,SARS-CoV的S和N基因的mRNA和蛋白质表达均显著减少。结论NSP1序列是SARS-CoV的RNAi策略的有效靶标,这些siRNAs很可能广泛地抑制了SARS-CoV亚基因组的合成及随后的蛋白质表达。

Objective To investigate new strategy for fighting severe acute respiratory syndrome （SARS）, a infectious disease caused by a novel coronavirus （SARS-CoV）, which spread over 30 countries in early 2003. Methods The effects of RNA interference （RNAi） on the infection and replication of this virus were investigated with 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyltetrazolium bromide （MTT）, plaque forming, Western blotting, and real-time PCR assays. Results The plasmid-derived siRNAs targeting non-structure protein 1 （ NSP1 ） sequence of 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 line. Compared with controls, the expressions of spike and nucleoprotein genes of the SARS-CoV at mRNA and protein level was significantly lower in siRNA-expressing cells at 1d post SARS-CoV infection. Condusion Our study provides strong evidence that NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the siRNAs might extensively inhibit the subgenome synthesis and subsequent proteins expression of SARS-CoV.