Mechanism Of The Interferon Stimulating Gene 15(ISG15) Regulation On Zika Virus Replication

Background&Aims:Zika virus(ZIKV)is an enveloped,single-stranded RNA virus which belong to the genus Flavivirus.ZIKV is an emerging pathogen worldwide and ZIKV infection can cause neurological diseases such as neonatal microcephaly and Guillain-Barre syndrome.There are no effective antiviral drugs and vaccines.Moreover,the pathogenic mechanism by which ZIKV causes nervous system damage is still not well characterized.Type I interferon(LFN)-mediated antiviral immune responses play an important role in the innate immune response.ZIKV has evolved multiple mechanisms to escape the host's anti-viral immune responses.However,the molecular mechanism by which ZIKV utilizes the host genes to escape the IFN anti-viral signaling pathway is not fully understood.ZIKV infection has been shown to stimulate interferon stimulated gene 15(ISG15)mRNA expression.The protein structure of ISG15 is similar to ubiquitin,so ISG15 is also called ubiquitin-like protein.ISG15 possess many biological functions including regulation on virus replication,tumor progression and immune response.Previous studies from our group have demonstrated that ISG15 promoted HBV and HCV replication.In this current study,we sought to explore the possible impact of ISG15 on ZIKV replication in A549 cells and its underlying mechanism.This study aimed the following aspects:(1)To test the effect of ISG15 on ZIKV replication;(2)To investigate whether ISG15 is regulated by type I interferon-mediated Jak/STAT signaling pathway.(3)To explore which form of ISG15(free form or ISG15 conjugated form)in the cells exerts its regulatory function on ZIKV replication,and(4)to study the mechanism by which ZIKV escapes the innate immune response.Results from these studies may provide new drug targets for the treatment of ZIKV infection.Methods:1 A549,Vero,Huh7,2fTGH and U5A cell lines were infected by ZIKV Cell samples were collected at different time points.ISG15 was over-expressed or knocked down in A549 cells infected with ZIKV,mRNA expression levels of selected genes including ISG15,IFN?,IFN?,IFN?,IFNy and ZIKV NS5 RNA were detected by real time PCR.The expression of ZIKV NS1 and capsid proteins were detected by western blot and IFC,respectively.2 ISG15 plasmid was transfected into ZIKV-infected A549,2fTGH and U5A cells with or without IFN? stimulation to investigate the effect of ISG15 on type I interferon(IFN)-mediated Jak/STAT signaling pathway.ZIKV NS5 RNA was evaluated by real time PCR.The phosphorylation levels of STAT1 and STAT2,the expressions of ISGs(MxA and IFIT1)in Jak/STAT signaling pathway and the expression level of ZIKV NS1 protein were detected by western blot.The activation status of interferon stimulated response element(ISRE)was measured by dual fluorescent reporter assay.3 ISG15 was knocked down by siRNA in A549 cells and then the cells were infected with ZIKV.The proteins level of ISGylation and ZIKV NS1 were detected by western blot.A549 cells were transfected with UbE1L siRNA(to inhibit ISGylation)following ZIKV infection,and the expression of NS5 RNA and NS1 protein of ZIKV were evaluated by real time PCR and western blot,respectively.Since ISG15 C-terminal LRLRGG sequence is essential to form ISGylation,we performed site-directed gene mutation from LRLRGG(wild-type)to LRLRAA(inactive mutant).Wild-type and mutant ISG15 plasmids were transfected into A549 cells and then the cells were infected with ZIKV.The expression of ZIKV NS5 RNA was analyzed by real time PCR.Results:1 ZIKV can infect and stably replicate in A549,Vero,Huh7,2fTGH and U5A cells.ZIKV infection induced the production of interferon ?,?,?,?,and ISG15.U5A cells are type I interferon receptor IFNAR-deficient cells.We found that ZIKV infection induced ISG15 production in U5A cells,indicating that the production of ISG15 was not completely dependent on type I interferon Jak-STAT pathway.Overexpression and knockdown of ISG15 did not affect the entry process of ZIKV into A549 cells.Overexpression of ISG15 promoted ZIKV replication in a dose-dependent manner,and knock down of ISG15 inhibited ZIKV replication.2 Knock down of ISG15 promoted the production of IFN?a and IFN?.In U5A cells(interferon receptor IFNAR-deficient cells),overexpression of ISG15 did not have effect on ZIKV replication.In contrast,in 2fTGH cells and A549 cells overexpression of ISG15 inhibited STAT1 and STAT2 phosphorylation,the activity of ISRE,and the expression of selected ISGs,and subsequently promoted ZIKV replication.3 Knock down of ISG15 in A549 cells and then the cells were infected with ZIKV to investigate the effect of ISG15/ISGylation on ZIKV replication.We found that ISGylation was weakened following ISG15 knock down and ZIKV replication was inhibited.Since UbE1L is the only activation enzyme for ISGylation,we performed knock down of UbE1L by specific siRNA and found that ISGylation level was significantly decreased while ZIKV was inhibited at both RNA and protein levels.Either ISG15 wild type or its inactive LRLRAA mutant were transfected into A549 cells infected with ZIKV.We found that wild type ISG15 promoted ZIKV replication and secretion,while the conjugation-inactive mutant had no effect on ZIKV replication and secretion.Conclusions:1 ZIKV infection stimulated the expression of ISG15 mRNA.However,the production of ISG15 was not completely dependent on the type I interferon signaling pathway.2 Over-expression of ISG15 promoted ZIKV replication in a dose-dependent manner;knock down of ISG15 significantly inhibited ZIKV replication.Overexpression or knock down of ISG15 did not affect the entry and endocytosis of ZIKV.ISG15 did not interact with ZIKV RNA,but bonded to ZIKV NS4A.3 ISG15 promoted the replication and secretion of ZIKV through negatively regulating type I interferon-mediated Jak/STAT signaling pathway.4 ISG15 enhanced ZIKV replication through ISGylation.