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Antisense downregulation of SARS-CoV gene expression in Vero E6 cells
1State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, People's Republic of China.
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 approximately 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 microM 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 microM or 0-30 microM.
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.
Insights
Antisense phosphorothioated oligonucleotides (PS-ODNs) effectively reduced expression of SARS-CoV structural proteins E, M, and N by over 90%. This demonstrates antisense knockdown as a promising strategy for SARS treatment and gene function studies.
Area of Science:
- Virology
- Molecular Biology
- Antisense Technology
Background:
- Severe acute respiratory syndrome (SARS) is caused by SARS-CoV, an enveloped RNA virus.
- Structural proteins E, M, and N are crucial for SARS-CoV's life cycle.
- Investigating methods to down-regulate these viral proteins is essential.
Purpose of the Study:
- To evaluate the efficacy of antisense phosphorothioated oligonucleotides (PS-ODNs) in down-regulating SARS-CoV structural protein expression.
- To assess the potential of antisense technology as a therapeutic strategy for SARS.
Main Methods:
- Vero E6 cells were transfected with constructs encoding SARS-CoV structural proteins (E, M, N) or fusion proteins with EGFP.
- Cells were treated with sequence-specific antisense PS-ODNs or control oligonucleotides.
- Gene expression levels were analyzed using RT-PCR.
Main Results:
- Six out of 26 tested antisense PS-ODNs achieved over 90% sequence-specific reduction in target gene expression.
- Down-regulation of fusion protein expression confirmed the antisense effect.
- The observed antisense effect was dose-dependent in Vero E6 cells.
Conclusions:
- Antisense PS-ODNs are effective in down-regulating SARS-CoV gene expression.
- Antisense knockdown presents a viable strategy for SARS treatment.
- This approach is suitable for studying SARS gene function in cellular models.
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