Small interfering RNA effectively inhibits the expression of SARS coronavirus membrane gene at two novel targeting sites.
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 unexplored 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.
Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. fish_wangying@sina.com
Ying
Y
Wang
Ying-Li
YL
Cao
Fan
F
Yang
Yun
Y
Zhang
Shu-Hui
SH
Wang
Li
L
Liu
D001483:Base Sequence; D000086862:Coronavirus M Proteins; D005814:Genes, Viral; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D016415:Sequence Alignment; D014763:Viral Matrix Proteins / Q000235:genetics*
D000086862:Coronavirus M Proteins; C000706237:M protein, SARS-CoV; D034741:RNA, Small Interfering; D014763:Viral Matrix Proteins