Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites

Small Interfering RNA Effectively Inhibits the Expression of SARS Coronavirus Membrane Gene at Two Novel Targeting Sites

Li Liu, Shu-Hui Wang, Fan Yang, Yun Zhang, Ying Wang, Yingli Cao

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.

2010

siRNA, SARS-CoV, membrane gene, RT-PCR, EGFP

DOI: 10.3390/molecules15107197

Molecules

MDPI AG

Organic chemistry

EN