| Background&Aims:Zika virus(ZIKV)is a single-stranded positive RNA virus transmitted by mosquito bites.ZIKV infection has attracted attention worldwide because it is closely associated with severe neurological complications in adults and microcephaly in infants.However,the pathogenesis of ZIKV has not been fully elucidated,and there are no specific antiviral drugs nor vaccines against the ZIKV.Therefore,it is essential to explore the pathogenesis of ZIKV and its interaction with the host,which is of great significance for preventing ZIKV infection and developing new antiviral strategies.MicroRNAs(miRNAs)are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level.MiRNAs have been reported to play a wide range of roles in the regulation of viral infection and related diseases.Although dysregulated expression of miRNAs has been reported,how these dysregulated miRNAs regulate ZIKV infection and their underlying mechanisms remain largely unexplored.The goal of this research is to dissect the role of differentially-expressed miRNAs caused by ZIKV infection in ZIKV replication and its underlying mechanism.Methods:1.Small RNA sequencing(sRNA-seq)was performed to identify differentially-expressed miRNAs in A549 cells with or without ZIKV infection.Three upregulated and three downregulated miRNAs were selected for further validation by quantitative real-time PCR(qPCR)in A549 and SH-SY5Y cells.To explore the effects of the selected miRNAs on ZIKV replication,we over-expressed miRNAs in A549 cells by miRNAs mimic transfection prior to ZIKV infection,and ZIKV NS5 mRNA was monitored by qPCR.2.MiR-103a-3p expression was upregulated or downregulated by transfection with miR103a-3p mimic or specific inhibitor in A549,THP-1,2fTGH and U5A cells prior to ZIKV infection to investigate the effects of miR-103a-3p on ZIKV replication and the phosphorylation of ERK,JNK and p38 MAPK.A549 cells were pretreated with JNK inhibitor SP600125,p38 MAPK inhibitor SB203580 or transfected with p38a siRNA prior to ZIKV infection to study the effect of blocking JNK pathway or p38 MAPK pathway on ZIKV replication.The potential target genes of miR-103a-3p were predicted by four algorithms,including miRanda,Targetscan,miRmap and picTar.One of the target genes,OTUD4,was further validated by mutation analysis with dual-luciferase reporter assay.OTUD4 was over-expressed by plasmid transfection or knockdown by siRNA prior to ZIKV infection to investigate the role of OTUD4 in ZIKV replication and p38 MAPK signaling pathway.qPCR was employed to monitor ZIKV NS5 mRNA and western-blot was performed to-detect ZIKV NS1 protein and the phosphorylation levels of ERK,JNK,p38 MAPK and HSP27.3.To determine the effect of OTUD4 on type-I interferon signaling pathway,we overexpressed OTUD4 in A549 or 293T cells with or without ZIKV infection and then treated with IFNβ.STAT1/STAT2 phosphorylation levels,ISRE activity,ISGs and IFNβexpression as well as ZIKV replication were analyzed by western blot,dual-luciferase reporter assay and qPCR,respectively.Immunoprecipitation was employed to identify the interaction between OTUD4 and various ZIKV proteins.A549 or 293T cells were transfected with ZIKV NS5 plasmid and then treated with IFNβ to determine the effects of NS5 over-expression on ISRE activity and ISGs expression.OTUD4 plasmid or siRNA was co-transfected with NS5 plasmid and then treated with IFNβ to investigate the effects of OTUD4 over-expression or knock-down on ISGs expression regulated by NS5 over-expression.Results:1.Thirty-five differentially-expressed miRNAs were identified in A549 cells following ZIKV infection through sRNA-seq analysis(p<0.05),of which fifteen were upregulated and twenty were downregulated.Some of those differentially-expressed miRNAs were validated by qPCR and the results were consistent with the sRNA-seq results.MiR-4454 or miR-493-3p over-expression significantly inhibited ZIKV replication,while miR-4093p or miR-103a-3p over-expression promoted ZIKV replication markedly.In addition,miR-1246 and miR-210-3p over-expression had no significant effect on ZIKV replication.2.MiR-103a-3p over-expression promoted ZIKV replication,while miR-103a-3p inhibitor inhibited ZIKV replication significantly.MiR-103a-3p showed no significant effect on adsorption nor endocytosis stage of ZIKV life cycle.Over-expression of miR103a-3p enhanced the phosphorylation levels of JNK and p38 MAPK while the phosphorylation level of ERK was not affected.SP600125 treatment significantly inhibited ZIKV replication but did not affect the stimulatory role of miR-103a-3p on ZIKV replication,indicating the promoting role of miR-103a-3p in ZIKV replication is independent of JNK signaling pathway.Either p38a silencing or SB203580 treatment inhibited ZIKV replication.SB203580 treatment attenuated the stimulating effect of miR-103a-3p on ZIKV replication,indicating that miR-103a-3p regulates ZIKV replication through p3 8 MAPK signaling pathway.A total of 177 potential target genes of miR-103a-3p were predicted by all the four different bioinformatics software.OTUD4 was one of the target genes involved in virus infection and p38 MAPK signaling pathway.Dual luciferase reporter assay confirmed that OTUD4 is a direct target gene of miR103a-3p.OTUD4 over-expression suppressed ZIKV replication and inhibited the activation of p38 MAPK signaling pathway.In contrast,silencing of OTUD4 stimulated ZIKV replication and activated the p38 MAPK signaling pathway.Furthermore,OTUD4 over-expression attenuated the stimulating effect of miR-103a-3p on ZIKV replication and activation of p38 MAPK signaling pathway.In addition,regulation of ZIKV replication by OTUD4 is partially dependent on p38 MAPK signaling pathway.3.OTUD4 over-expression promoted the phosphorylation levels of STAT1/STAT2,ISRE activity and ISGs expression induced by ZIKV infection or IFNβ stimulation,and enhanced IFNβ expression to promote the anti-ZIKV effect of IFNβ.OTUD4 specifically interacted with ZIKV NS5 protein,which attenuated the inhibitory effect of NS5 on ISGs expression induced by IFNβ stimulation.Conclusions:1.Thirty-five differentially expressed miRNAs were identified by sRNA-seq analysis in ZIKV-infected A549 cells.One of the validated up-regulated miRNAs,miR-103a-3p,significantly stimulated ZIKV replication.2.MiR-103a-3p stimulated ZIKV replication by targeting OTUD4 to activate p38 MAPK signaling pathway.3.OTUD4 over-expression inhibited ZIKV replication through promoting type Ⅰinterferon signaling.OTUD4 specifically interacted with ZIKV NS5 protein to attenuate the inhibitory effect of NS5 on type Ⅰ interferon signaling. |
