Analysis Of MiRNAs And Lead Compounds That Inhibit Enterovirus71Replication

Hand, foot, and mouth disease (HFMD) outbreaks have been reported in many area, especially in Aria. Enterovirus71(EV71) is the major causative agent of HFMD in children and infants, its infection usually accompanied with severe neurological complication which causes high mortality. In recent years, the molecular biology study of EV71and the progress of antiviral agents/methods development provide us new strategies for the treatment and prevention of EV71infection.Micro RNAs (miRNAs) is a kind of small single strand non-protein-coding RNAs of lengths about22nucleotides. miRNAs can partially complementary to3’untranslated region(3’-UTR) elements in the mRNA, mediate posttranscriptional repression through inhibiting translation or triggering degradation. miRNAs have been found playing critical roles in a wide variety of biological processes, including the modulation of intricate host-pathogen interaction networks. Here we investigate the role of miRNAs in EV71virus replication.First, each segment of EV71virus genome was inserted to the pMIR vector and the luciferase expression was assayed to identify the target gene of putative miRNA.The reported gene expression of the cells transfected with the vector containing5’-UTR was significantly downregulated.In order to investigate the miRNA target region of5’-UTR gene,we fractured the gene into3subfragments with the length about260bp, and found luciferase expression of pMIR vector which contains5’-UTR-1subfragments was downregulated1/4compared with control.Then we screened the miRNAs that may target to5’-UTR-1using online analysis programs,2candidate miRNAs were selected and synthesized mimics.The results showed that the expression of the reporter gene was downregulated by these two miRNAs. Futhermore, we investigated the effect of miR-373and miR-542-5p on viral replication.miR-373and miR-542-5p was used to address the replication of EV71virus in RD cells. Western blot and real-time PCR test were performed and the results showed that, miR-373and miR-542-5p can suppress EV71virus replication.The other part of this article examined the effect of6lead compounds that inhibits EV71infection. The EV71VP1capsid protein expression levels were tested by Western blot, which indicated that the compound was able to inhibit EV71replication. The cytotoxic activity of the compound was evaluated against Rhabdomyosarcoma (RD) cells in vitro by MTT assay. The results showed that the compound have low toxicity with a CC50of0.0726μg/μL. After incubated with the compound at a concentration of0.01μg/μL for48hours, the levels of EV71vpl mRNA in RD cells was decreased by76.83±2.47%.When methyl3,4-dihydroxyphenylacetatate was passively administered to one-day-old suckling mice which had been infected with EV71virus,their subsequent growth were better than positive control.And methyl3,4-dihydroxyphenylacetatate can inhibit EV71virus proliferation in suckling mice.These findings suggested that methyl3,4-dihydroxyphenylacetatate is suitable as a lead compound for antiviral therapies against EV71, which may contribute to the discovery of new drugs.Two pentapeptides were desigened to inhibit EV71virus3Cpro. A compound, P010157,was identified as a potent inhibitor by Western blot and real-time PCR while showing a low level of cytotoxicity(CC50～155.7378μg/μL).This paper gives us an understanding of the functions of human miRNAs and compounds in inhibiting EV71virus replication,and provides us a new insights into the therapy of EV71virus infection.