The Role Of MiR-133a-3p In ZIKV Replication And Its Underlying Mechanism

BackgroundZika Virus(ZIKV)can be transmitted to humans through mosquitoes.Most infected people are asymptomatic,but in severe cases ZIKV infection can cause neonatal microcephaly and Guillain-Barre syndrome in adults.There are currently no vaccines nor antivirals specific for ZIKV infection.Therefore,it is very important to study the pathogenesis of ZIKV and to explore the possible antiviral drug targets.(1)A large number of studies have shown that viral infections can change the expression levels of microRNAs(miRNAs);meanwhile,by inhibiting the expression of certain target proteins,miRNAs can activate the host type ?interferon signaling pathway to restrict viral infection.Previous reports have shown that miR-133a-3p could significantly inhibit dengue virus replication.We also found the expression level of miR-133a-3p was increased inZIKV-infected A549 cells,but its biological significance,especially the anti-ZIKV effect has not been reported.Therefore,it is of great importance to investigate the effect of miR-133a-3p on ZIKV replication and its underlying mechanism.(2)As a negative regulator of IFN signaling,IRF2(Interferon regulatory factor 2)competes with IRF1 for the ISRE sequence at the promoter regions of the interferon-stimulated genes(ISGs)and the IRF-E sequence of the IFN?promoter region,thereby inhibiting the production of IFN? and the type I interferon signaling pathway to promote virus replication.In addition,studies have shown that IRF2 regulates viral replication by regulating FAM111A and RFC3.However,how IRF2 regulates ZIKV replication remains to be clarified.Purpose(1)To study the role of increased expression of miR-133a-3p in ZIKV replicatioin and its potential mechanism;(2)To explore the role of IRF2 in ZIKVreplication and its underlying mechanism;(3)To clarify the molecular mechanism of miR-133a-3p and IRF2 in regulating ZIKV replicationContents(1)To study the effect of ZIKV infection on miR-133a-3p expression;(2)To study the mechanism on how miR-133a-3p affects ZIKV replication through regulation of Jak/STAT signaling pathway;(3)To confirm IRF2 is one of the target genes of miR-133a-3p;(4)To clarify the role ofIRF2 in regulation of Jak/STAT signaling and ZIKV replication and the underlying mechanism.Methods(1)A549 cells were infected with ZIKV and the cells were harvested at different time points.The expression levels of miR-133a-3p were determined by RT-qPCR;(2)miR-133a-3p was over-expressedin A549 cells followed by ZIKV infection and the replication of ZIKV was detecetd by RT-qPCR and Western blots;(3)miR-133a-3p was over-expressed in A549 cells followed by ZIKV infection or treatment with polyI:C and the expression levels of ZIKV RNA,IFN?,IFN?,ISG15,and IFIT1 were determined by RT-qPCR;miR-133a-3p mimic or negative control was co-transfecetd with ISRE-luc and pRL-TK reporter plasmids into A549 cells and the ISRE activity was monitored by dual-luciferase reporter assay.These results will clarify the effect of increased expression of miR-1 33a-3p on ZIKV replication and IFN signaling pathway;(4)miR-133a-3p mimic was transfected into A549cells and IRF2 expression level was detected byRT-qPCR and Western blots.miR-133a-3p mimic or negative control was co-transfected with pmiR-IRF2-WT or pmiR-IRF2-Mut plasmids to confirm that there exists miR-133a-3p binding site at 3'UTR of IRF2;(5)IRF2 was knocked-down in A549,2FTGH and U5A cells followed by ZIKV infection in the presence or absence of IFN-? and the total RNA was extracted.The expression levels of ZIKV RNA,IRF2,IFN?,ISG15 and IFIT1 were determined by RT-qPCR to clarify the role of IRF2 in ZIKV replication and IFN signaling;(6)FAM111A or RFC3 was knocked-down respectively in 2FTGH,U5A and IRF2-overexpressed cells followed by ZIKV infection.ZIKV replication was detected by RT-qPCR to clarify whether IRF2 regulates ZIKV replication through FAM111A or RFC3.Results(1)The expresion level of miR-133a-3p was increased significantly in ZIKV-infected A549 cells;(2)miR-133a-3p inhibits ZIKV replication and activates the Jak/STAT signaling pathway;(3)Knock-down of IRF2 activates type-I IFN signaling but does not play a major role in inhibiting ZIKV replication;(4)IRF2 activates FAM111A expression to enhance the host restriction effect of RFC3 on ZIKV infection.Conclusions(1)The expression level of miR-133a-3p is significantly increased following ZIKV infection.Increased expression of miR-133a-3p inhibits ZIKV replication through activativation of the type I interferon signaling pathway leading to the increased expression of antiviral interferon-stimulated genes(ISGs).(2)Knock-down of IRF2 activates the type I interferon signaling pathway,which does not play a leading role in the regulation of ZIKV replication.Instead,IRF2 activates FAM111A expression to enhance the host restriction effect of RFC3 on ZIKV infection.