Effect Of Interferon-stimulated Gene 12a And Interferon-stimulated Gene16 On Zika Virus Replication And The Underlying Mechanism

BackgroundZika virus(ZIKV)is a mosquito-borne virus that can cause microcephaly in neonates and neuropathy,such as Guillain-Barre syndrome in adults.Currently,neither vaccine nor specific treatment is available.Type ? Interferon(interferon,IFN)is one of the most important molecules of the host innate immune system and plays an important role in anti-ZIKV infection.After binding to the receptor,type ? IFN activates the JAK/STAT signaling pathway to stimulate the expression of a series of interferon-stimulated genes(ISGs)to exert its antiviral and immunomodulatory effects.Although ISG12 and ISG16 are ISGs and belong to the same family,they have opposite role in HCV replication.These data indicate that ISG12 and ISG16 are related to virus infection,but their effect on Zika virus replication has not been reported,and the underlying mechanism needs to be further studied.ObjectiveTo study the role of ISG12a/ISG16 in ZIKV replication and the underlying mechanismMethods1.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited by plasmid transfection or siRNA silencing,and the cells were infected with ZIKV.The role of ISG12a/ISG16 in ZIKV replication was evaluated by real-time PCR and Western Blot;2.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited by plasmid transfection or siRNA silencing,and the cells were treated with IFN?.The role of ISG12a/ISG16 in the production of endogenous IFN? and activation of JAK/STAT signaling pathway was evaluated by the expression levels of IFN? and key molecules in the IFN?-producing pathways(real-time PCR),phosphorylation of STAT1(p-STAT1,Western Blot),interferon-stimulated response element(ISRE)activity(dual-luciferase reporter assay),and ISGs expression(real-time PCR);3.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited by plasmid transfection or siRNA silencing,and the cells were infected with ZIKV followed by IFN? treatment.The role of ISG12a/ISG16 in IFN? anti-ZIKV was assessed by real-time PCR.Results1.Both ZIKV infection and IFN? treatment stimulated ISG12a and ISG16 expression in A549 cells.Overexpression of ISG12a or ISG16 inhibited ZIKV replication while silencing of ISG12a or ISG16 stimulated ZIKV replication.2.ISG12a or ISG16 stimulated the production of endogenous IFN? through increased expression of RIG-I?TLR3 and MDA5 and activated the downstream JAK/STAT signaling pathway as shown by the increased p-STAT1 level,enhanced ISRE activity and over-expressed ISGs to inhibit ZIKV replication.3.ISG12a or ISG16 enhanced the anti-ZIKV effect of exogenous IFN ?.ConclusionISG12a or ISG16 inhibits ZIKV replication through increased expression of endogenous IFN ? and activation of JAK/STAT signaling pathway.In addition,both ISG12a and ISG16 enhance the anti-ZIKV effect of exogenous IFN?.These results indicate that ISG12a and ISG 16 play an important role in the innate immune response against ZIKV infection.Background Zika virus(ZIKV)is a mosquito-borne virus that can cause microcephaly in neonates and neuropathy,such as Guillain-Barre syndrome in adults.Currently,neither vaccine nor specific treatment is available.Type I Interferon(interferon,IFN)is one of the most important molecules of the host innate immune system and plays an important role in anti-ZIKV infection.After binding to the receptor,type I IFN activates the JAK /STAT signaling pathway to stimulate the expression of a series of interferon-stimulated genes(ISGs)to exert its antiviral and immunomodulatory effects.Although ISG12 and ISG16 are ISGs and belong to the same family,they have opposite role in HCV replication.These data indicate that ISG12 and ISG16 are related to virus infection,but their efTcct on Zika virus replication has not been reported,and the underlying mechanism needs to be further studied.Objective To study the role of ISG12 a / ISG16 in ZIKV replication and the underlying mechanism Methods1.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited by plasmid transfection or siRNA silencing,and the cells were infected with ZIKV.The role of ISG12a/ISG16 in ZIKV replication was evaluated by real-time PCR and Western Blot;2.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited by plasmid transfection or siRNA silencing,and the cells were treated with IFN?.The role of ISG12a/ISG16 in the production of endogenous IFN? and activation of JAK /STAT signaling pathway was evaluated by the expression levels of IFN? and key molecules in the IFN?-producing pathways(real-time PCR),phosphorylation of STATl(p-STATl,Western Blot)5 interferon-stimulated response element(ISRE)activity(dual-luciferase reporter assay),and ISGs expression(real-time PCR);3.The expression level of ISG12a/ISG16 in A549 cells was up-regulated or inhibited byplasmid transfection or siRNA silencing,and the cells were infected with ZIKV followed by IFN? treatment.The role of ISG12a/ISG16 in IFN? anti-ZIKV was assessed by real?time PCR.Results1.Both ZIKV infection and IFN? treatment stimulated ISG12 a and ISG16 expression in A549 cells.Overexpression of ISG12 a or ISG16 inhibited ZIKV replication while silencing of ISG12 a or ISG16 stimulated ZIKV replication.2.ISG12 a or ISG16 stimulated the production of endogenous IFN? through increased expression of RIG-I?TLR3 and MDA5 and activated the downstream JAK /STAT signaling pathway as shown by the increased p-STATl level,enhanced ISRE activity and over-expressed ISGs to inhibit ZIKV replication.3.ISG12 a or ISG16 enhanced the anti-ZIKV effect of exogenous IFN p.Conclusion ISG12 a or ISG16 inhibits ZIKV replication through increased expression of endogenous IFN Pand activation of JAK/STAT signaling pathway.In addition,both ISG 12 a andISG16enhance the anti-ZIKV effect of exogenous IFN?.These results indicate that ISG12a and ISG 16 play an important role in the innate immune response against ZIKV infection.