Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.
Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.
Qin
Zhao-ling
ZL
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Zhao
Ping
P
Zhang
Xiao-lian
XL
Yu
Jian-guo
JG
Cao
Ming-mei
MM
Zhao
Lan-juan
LJ
Luan
Jie
J
Qi
Zhong-tian
ZT
D002460:Cell Line; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*
D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins