Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA.
Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.
Ni
Bing
B
Institute of Immunology PLA, Third Military Medical University, Chongqing, China. nibing@vip.163.com
Shi
Xinfu
X
Li
Yang
Y
Gao
Wenda
W
Wang
Xiliang
X
Wu
Yuzhang
Y
D000818:Animals; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000502:physiology; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D034741:RNA, Small Interfering; D017361:Viral Nonstructural Proteins