RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells.
- Author:
Xu-Dong ZHU
1
;
Ying DANG
;
Yi FENG
;
Tao LI
;
Pei-Tang HUANG
Author Information
1. Beijing Institute of Biotechnology, Beijing 100071, China. xd_zhu@263.net
- Publication Type:Journal Article
- MeSH:
Cells, Cultured;
Humans;
Plasmids;
RNA Interference;
RNA, Messenger;
metabolism;
RNA, Small Interfering;
genetics;
RNA, Viral;
metabolism;
Ribonuclease III;
physiology;
SARS Virus;
genetics
- From:
Chinese Journal of Biotechnology
2004;20(4):484-489
- CountryChina
- Language:Chinese
-
Abstract:
SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
