Antisense downregulation of SARS-CoV gene expression in Vero E6 cells.
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.
Shi
Yi
Y
State 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.
Luo
Haifeng
H
Jia
Jie
J
Xiong
Jie
J
Yang
Dehua
D
Huang
Bing
B
Jin
Youxin
Y
D000818:Animals; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells
D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins