Potent and specific inhibition of SARS-CoV antigen expression by RNA interference.
BACKGROUND
Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression.


METHODS
The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.


RESULTS
The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.


CONCLUSIONS
Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.
Tao
Peng
P
The Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
Zhang
Jun
J
Tang
Ni
N
Zhang
Bing-qiang
BQ
He
Tong-chuan
TC
Huang
Ai-long
AL
D000956:Antigens, Viral / Q000235:genetics*; D002478:Cells, Cultured; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D034622:RNA Interference; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000276:immunology; D045169:Severe Acute Respiratory Syndrome / Q000628:therapy
D000956:Antigens, Viral; D019590:Nucleocapsid Proteins; D049452:Green Fluorescent Proteins