Inhibition of Replication and Infection of Severe Acute Respiratory Syndrome-Associated Coronavirus With Plasmid-Mediated Interference RNA 

Authors:
Bing Ni  1 , Xinfu Shi, Yang Li, Wenda Gao, Xiliang Wang, Yuzhang Wu
In:
Source:
Publication Date: (2005)
Issue: 10: 4
Research Area:
Respiratory Research
Cells used in publication:
Vero
Species: monkey
Tissue Origin: kidney
Culture Media:
Platform:
Nucleofector™ I/II/2b
Experiment

To study the effects of siRNAs targeting the NSP1-EGFP fusion gene in vitro, 1 µg psiRNA and 1.5 µg pEGFP-N1/NSP1 were cotransfected into Vero E6 cells by an amaxa / Nucleofector electroporation device.

Abstract

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.