Identification And Verification Of Cellular Targets For Anti-EV71Drugs

Enterovirus71(enterovirus type71, EV71) is one of the serious public healthproblems in the world since the first reported in1974, and the infection has beencaused in the worldwide many times. However, there is no effective method toprevent and resist EV71; the main problem is due to the smaller viral genomes and thehigher mutation.Recent studies have shown that inhibiting the function of certain proteins in thehost cells can block virus infection. Compared with the viral genome, there are moregenes related with virus replication in the human genome sequence, and some studiesalso proved that inhibiting host gene expression related with EV71replication canblock the infection of EV71virus.Therefore, the aim of this project is to discover thepotential anti-EV71drug targets in the host cells and viruses by the bioinformaticsmethodand to identify and validate the new potential targets of viruses and host cellsfor anti-EV71drugs in vitro and in vivo by using antisense oligonucleotidestechnology, which lay the foundation for the screening of new antiviral drugs.Combined with gene expression profiles of human anti-EV71(the profilespectrum number GSE16358) and human protein interaction networks (HPRD), thisstudy use subnet identification method to design a core subnet containing more than100nodes then select important nodes in the topology. Finally, we have usedbioinformatics analysis methods to filter out the genes of CDK1, PIN1, CSNK2A2,CSNK2B, VIM, MAPK3, PRNP, YWHA2, CREBBP, KHDRBS1, ESR1andMAPK8, which may become the potential anti-EV71drug targets. According to thevarious genes in GeneBank, we developed computer aided design based on thesecondary structure prediction and got five of ASODNs（Antisense oligonucleotides）targeting the above all genes. At the same time, five antisense oligonucleotidesequences EV1, EV2, EV3, EV4, and EV5for EV71genome were designed using ofbioinformatics methods. The effective sequence were screened through the influenceof EV71replication by CPE （cytopathologetic ettect） analysis, fluorescencequantitative PCR and western blotting technique in vitro and in vivo. This studydetermined that the antisense sequence of EV5targeting to EV71and the ASODN ofCDK1had good anti-EV71virus activity; meanwhile, we found that CDK1andCSNK2A2may become new anti-EV71drug targets.Due to multi-target combination therapy has gradually become a hot topic in the anti-EV71treatment; we filtered out the combined treatment group of better effects onthe basis of preliminary works, which provides a new way of the clinical combinedantiviral therapy.