The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

摘要

BackgroundEnterovirus 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.MethodsUnmodified 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.ResultsTransfection 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.ConclusionSequences 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.