Antisense downregulation of SARS‐CoV gene expression in Vero E6 cells

Shi, Yi; Luo, Haifeng; Jia, Jie; Xiong, Jie; Yang, Dehua; Huang, Bing; Jin, Youxin

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS‐CoV). It is an enveloped, single‐stranded, plus‐sense RNA virus with a genome of ∼30 kb. The structural proteins E, M and N of SARS‐CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down‐regulated using an antisense technique. METHODS: Vero E6 cells were transfected with plasmid constructs containing exons of the SARS‐CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS‐ODN, 20mer) or a control oligonucleotide by addition to the culture medium. RESULTS: Among a total of 26 antisense PS‐ODNs targeting E, M and N genes, we obtained six antisense PS‐ODNs which could sequence‐specifically reduce target genes expression by over 90% at the concentration of 50 µM in the cell culture medium tested by RT‐PCR. The antisense effect was further proved by down‐regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS‐ODNs in a range of 0–10 µM or 0–30 µM. CONCLUSIONS: The antisense PS‐ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system. Copyright © 2004 John Wiley & Sons, Ltd.