Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71.
Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72-79]. The viral inhibitory effects lasted 72 h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3D(pol) region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.
Tan
Eng Lee
EL
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, Singapore 117597, Singapore.
Tan
Theresa May Chin
TM
Chow
Vincent Tak Kwong
VT
Poh
Chit Laa
CL
D045744:Cell Line, Tumor; D003588:Cytopathogenic Effect, Viral; D029821:Enterovirus A, Human / Q000502:physiology*; D004769:Enterovirus Infections / Q000628:therapy* / Q000821:virology; D018390:Gene Targeting; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication
D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins