The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs.
BACKGROUND
Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71.


METHODS
Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2'-modified (2'-O-methylation or 2'-fluoro modification) siRNAs were designed to target highly conserved 5' untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs.


RESULTS
Transfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5' UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2'-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts.


CONCLUSIONS
Sequences were identified within the highly conserved 5' UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2'-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.
Department of Pediatrics, Xijing Hospital, Fourth Military Medical University, 15th Changlexi Road, Xi'an 710032, PR China.
Jun-Xia
JX
Deng
Xiao-Jing
XJ
Nie
Ying-Feng
YF
Lei
Chao-Feng
CF
Ma
Dong-Liang
DL
Xu
Biao
B
Li
Zhi-Kai
ZK
Xu
Guo-Cheng
GC
Zhang
D020121:5' Untranslated Regions / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D029821:Enterovirus A, Human / Q000737:chemistry / Q000235:genetics*; D004769:Enterovirus Infections / Q000235:genetics / Q000628:therapy / Q000821:virology; D006801:Humans; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000821:virology; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*
D020121:5' Untranslated Regions; D034741:RNA, Small Interfering