The Role Of CircRNA(Circular RNA) In Zika Virus Replication And Its Underlying Mechanism

Background and Aims:Zika virus(ZIKV)is a single-stranded RNA virus belonging to the genus Flavivirus and is mainly transmitted by the Aedes mosquito.ZIKV is of great concern worldwide because ZIKV infection in pregnant women can cause microcephaly in newborns.CircRNAs(Circular RNAs)are a class of non-coding RNAs with a specific closed-loop structure.CircRNAs have been reported to be involved in a variety of physiopathological processes,including viral replication,antiviral immune responses,and infectious disease processes.Many studies have shown that circRNAs are dysregulated in host cells after viral infection and that circRNAs can act as microRNAs(miRNAs)sponges to regulate the expression of downstream target genes.However,there are few reports on whether circRNAs are also dysregulated in ZIKV-infected host cells and how circRNAs regulate ZIKV replication and the specific mechanisms.Therefore,in this study,based on the differentially expressed circRNAs screened by RNA-sequencing after ZIKV infection,we investigated the effect and mechanism of circRNA on ZIKV replication.Methods:Expression changes of hsa＿circ＿0007321 and hsa＿circ＿0000290 after ZIKV infection were verified by qRT-RCP.The hsa＿circ＿0007321 siRNA or hsa＿circ＿0000290 siRNA was transfected into A549 and U251 cells and then infected with ZIKV to study the effect of hsa＿circ＿0007321 and hsa＿circ＿0000290 on ZIKV replication.The localization of hsa＿circ＿0007321 and hsa＿circ＿0000290 in cells was confirmed by nucleoplasmic localization assay.Interaction of hsa＿circ＿0007321 with miR-492 was verified by RNA pull down and dual luciferase reporter gene assays.hsa＿circ＿0007321 siRNA or miR-492 mimic or miR-492 inhibitor were transfected into A549 cells to investigate the effects of hsa＿circ＿0007321/miR-492 on ZIKV replication,inflammatory cytokine IL6/8 expression,total IκBa protein levels and NF-κB p65 phosphorylation levels.The possible target genes of miR-492 were analyzed by bioinformatics methods,and the effect of miR-492 on NFKBID levels was verified by qRT-PCR and Western blot,and the targeting effect of miR-492 on NFKBID was verified by dual luciferase reporter gene assay.NFKBID siRNA was transfected into A549 cells to investigate the effects of NFKBID on ZIKV replication,inflammatory cytokine IL6/8 expression,total IκBa protein levels and NF-κB p65 phosphorylation levels.Results:Expression of hsa＿circ＿0007321 was significantly down-regulated after infection with ZIKV and hsa＿circ＿0000290 was significantly up-regulated after infection with ZIKV.Knockdown of either hsa＿circ＿0007321 or hsa＿circ＿0000290 expression levels inhibited ZIKV replication.The results of nucleoplasmic localization showed that hsa＿circ＿0007321 was mainly localized in the cytoplasm and hsa＿circ＿0000290 was mainly localized in the nucleus.hsa＿circ＿0007321 acted as a miRNA "sponge" to adsorb miR-492,and miR-492 targeted to NFKBID.Overexpression of mR-492 or NFKBID silencing activates the NF-κB signaling pathway,promotes inflammatory cytokine expression,and inhibits ZIKV replication.Conclusion:Zika virus infection induces down-regulation of hsa＿circ＿0007321 expression and up-regulation of hsa＿circ＿0000290 expression in host cells.hsa＿circ＿0007321 acts as a competitive endogenous RNA(ceRNA)that plays a role in regulating ZIKV replication through the miR-492/NFKBID NF-κB signaling pathway.