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 β expression. Thus, our results indicate that SARS-CoV M gene specific siRNA might function in a sequence-dependent manner.
【저자키워드】 SARS-CoV, RT-PCR, siRNA, eGFP, Membrane gene, 【초록키워드】 viral infection, coronavirus, Gene Expression, SARS-CoV, qRT-PCR, interferon, RT-PCR, RNA, Protein, Region, siRNA, membrane, novel, information, expression, Quantitative, Critical, nucleotide, viral genome, isolates, Small interfering RNA, sequence, SARS-CoV infection, upregulation, nucleotide substitution, targeting, M gene, Effect, isolate, tested, analyzed, blocked, conserved, significantly, occur, demonstrated, SARS-CoV M, downregulated, Site, RNA molecule, post-transcriptionally, downregulate, semi-quantitative,