Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.
A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.
Meng
Bo
B
Key Lab of Brain Functional Genomics, MOE & STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai, China 200062.
Lui
Yue-woon
YW
Meng
Shi
S
Cao
Changxiu
C
Hu
Yinghe
Y
D000818:Animals; D015967:Gene Expression Regulation, Viral; D051379:Mice; D016254:Mutagenesis, Insertional; D041681:NIH 3T3 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*; D000086082:Viroporin Proteins
C501689:E protein, SARS coronavirus; D034741:RNA, Small Interfering; D014759:Viral Envelope Proteins; D000086082:Viroporin Proteins; D012324:RNA-Dependent RNA Polymerase