The Study On Mechanism Of VX-11e Inhibiting Zika Virus Replication

Background and ObejectiveZika virus(ZIKV)is one of several emerging insect-borne viruses and it was first discovered in Zika Forest in Uganda in 1947.Also,it was the cause of encephalitis and fetal microcephaly outbreaks in Brazil and Puerto Rico.Zika virus can be transmitted by Aedes mosquitoes.Unlike other mosquito-borne flaviviruses(FVs),Zika virus unique characteristics,which can be transmitted through sexual transmission or even cause continuous transmission(1 to 6 months).The Zika virus that was circulating in the Americas in 2015 had triggered a global public health emergency.It is also predicted that in the Americas,4 million clinical cases caused by Zika virus can occur every year.Placenta and blood-brain barrier endothelial cells(ECs)usually block the mixing of maternal and fetal blood and limit the entry of some substances into the neuronal compartment of adults and fetuses.The Zika virus can pass through these cells and infect both fetuses and mothers.Infected cells include neural stem cells,astrocytes,neural progenitor cells,microglia,and Hofbauer cells.According to reports,phylogenetic tree analysis found that dengue viruses(DENVs),which are also commonly transmitted in humans and mosquitoes,have striking homology with Zika virus.The reasons for the recent outbreaks of epidemics and the surge in serious diseases are still unclear,and there is an urgent need for experimental systems to address these key scientific issues.Recent studies have found that Zika virus infection is related to the mitogen-activated protein kinase(MAPK)signaling pathway,and the MAPK-ERK signaling pathway is still a research hotspot.Therefore,our research group mainly studies the mechanism of ERK2 inhibitor VX-11e inhibiting the replication and proliferation of Zika virus,which may provide new clues for the research of drug treatment of clinical Zika virus disease.Methods1.The impact of VX-11e on Zika virus replicationVero cells had been infected after infecting and titering determination of Zika virus in Vero cells and HBMECs.Real-time PCR was used to detect the viral copy numbers in the cells treated with different concentrations of VX-11e,at the same time,the expression level of extracellular regulated protein kinase(ERK)and ribosomal S6 protein kinase(RSK)were determined.2.The impact of RSK on Zika virus replicationThe expression levels of RSK and ERK were detected after silencing RSK and ERK gene,and the expression of RSK and ERK were down regulated after transfected siRNA.The qPCR was used to detect the replication folds of Zika virus,and the relationship between viral replication and RSK and ERK expression were determined.3.Construction of Zika virus NS5 expression vectorThe Zika virus NS5 expression vector was constructed by using eukaryotic expression vector pEGFP-N1.Result1.Zika virus can be amplified in HBMECs,which is consistent with recent reports.In particular,ZIKV can infect HBMECs,explaining why ZIKV can cause nervous system infection.2.VX-11e has no toxicity on HBMECs and Vero cells within a certain concentration range,and has obvious inhibitory effect on Zika virus in a concentration-dependent manner.VX-11e does not affect the adhesion of zika virus to the cells,but inhibits the replication of the virus in cells.3.VX-11e can suppress RSK expression at the mRNA and protein levels4.Preliminary experimental results suggest that inhibition of RSK expression has an inhibitory effect on viral replication5.Successful construction of pEGFP-NS5 protein expression vector.6.VX-11e inhibit Zika virus produced by transfection of Zika virus full-length clone Z0E2-GFP,and when Z0E2-mute did not express NS5 protein after mutation,the inhibitory effect disappeared,and pEGFP-NS5 expression vector was added to express NS5.After the protein exerted its function,the inhibitory effect was restored.ConclusionVX-lle inhibits the expression of RSK in cells,thereby affecting the NS5 protein of Zika virus,and finally inhibits the replication of Zika virus in cells.Through the research in this paper,it provides new clues for the researches of drug therapy for clinical Zika virus disease.