Antisense downregutation of SARS-CoV gene expression in Vero E6 cells

摘要

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 similar to30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and vital 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. N-and N 0 a. genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 mu,M in the cell culture medium tested by RT-PCR. The antisense effect was 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 muM or 0-30 muM.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 (C) 2004 John Wiley Sons, Ltd.