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Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites
Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
* Author to whom correspondence should be addressed.
Received: 1 September 2010; Accepted: 15 September 2010 / Published: 18 October 2010
Abstract: Small interfering RNA (siRNA) is a class of duplex RNA molecules of 21-25 nt nucleotides in length functioning post-transcriptionally to downregulate targeted gene expression. The membrane (M) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is highly abundant during viral infections and is a critical element for viral assembly. Nucleotide substitution in the viral genome occurs frequently during SARS-CoV infection. In the current study, we analyzed the M gene sequences derived from 15 SARS-CoV isolates and uncovered six nucleotide substitutions among these isolates. Interestingly, these nucleotide substitutions are all located at the 5’ half of the M gene. Based on this information and previous reports, we created two novel siRNAs targeting two unexploited and well conserved regions in the M gene. The effects of these two siRNAs were tested by semi-quantitative RT-PCR and EGFP-M fusion gene expression. The results demonstrated that both siRNAs effectively and specifically blocked the targeted gene expression. Real time quantitative RT-PCR (qRT-PCR) revealed that siRNA targeting the 3’ half of the M gene (si-M2) induced more potent inhibition than that targeting the 5’ half (si-M1). Both si-M1 and si-M2 significantly downregulated M gene mediated upregulation of interferon b expression. Thus, our results indicate that SARS-CoV M gene specific siRNA might function in a sequence-dependent manner.
Keywords: siRNA; SARS-CoV; membrane gene; RT-PCR; EGFP
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Wang, Y.; Cao, Y.-L.; Yang, F.; Zhang, Y.; Wang, S.-H.; Liu, L. Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites. Molecules 2010, 15, 7197-7207.
Wang Y, Cao Y-L, Yang F, Zhang Y, Wang S-H, Liu L. Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites. Molecules. 2010; 15(10):7197-7207.
Wang, Ying; Cao, Ying-Li; Yang, Fan; Zhang, Yun; Wang, Shu-Hui; Liu, Li. 2010. "Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites." Molecules 15, no. 10: 7197-7207.