title: [RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells].
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.
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China. xd_zhu@263.net
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Zhu
authors.forename: Ying
authors.initials: Y
authors.lastname: Dang
authors.forename: Yi
authors.initials: Y
authors.lastname: Feng
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Pei-Tang
authors.initials: PT
authors.lastname: Huang
mesh_terms: D002478:Cells, Cultured; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism*; D043244:Ribonuclease III / Q000502:physiology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral; D043244:Ribonuclease III