Akerström Sara, Mirazimi Ali, Tan Yee-Joo
Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden.
Antiviral Res. 2007 Mar;73(3):219-27. doi: 10.1016/j.antiviral.2006.10.008. Epub 2006 Nov 7.
The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19 nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19 nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.
严重急性呼吸综合征冠状病毒（SARS-CoV）基因组有14个潜在的开放阅读框（ORF）。第一个ORF由全长基因组mRNA翻译而来，其余的ORF则由8个亚基因组RNA（sgRNA）翻译而来。在本研究中，我们设计了靶向sgRNA 2、3和7的小干扰RNA（siRNA），并测试了它们在沉默靶向sgRNA翻译的蛋白质方面的效率和特异性。我们的结果表明，siRNA 7可以抑制sgRNA 7（显示19/19个核苷酸（nt）匹配）和sgRNA 8（显示18/19 nt匹配）；但是，它不抑制全长基因组mRNA（显示17/19 nt匹配）。总体而言，每个siRNA都可以抑制靶向的sgRNA，而不影响全长基因组mRNA或其他显示两个或更多nt错配的sgRNA。因此，可以设计siRNA，以便在病毒感染期间敲低靶向sgRNA的病毒蛋白表达，从而能够描绘出单个病毒蛋白对病毒感染的贡献。当用SARS-CoV感染表达siRNA 2、3或7的Vero E6细胞时，观察到子代病毒产量显著降低。间接免疫荧光分析表明，在表达每种siRNA的感染细胞中，分别存在S、3a和7a的特异性沉默，但核衣壳蛋白的表达未受影响。因此，我们的数据表明，辅助蛋白，即3a和7a，可能在SARS-CoV的复制周期中发挥重要作用。
