| Background and Aims:Zika virus(ZIKV)is a mosquito-borne RNA virus belonging to the genus Flavivirus of the Flaviviridae family.It was once declared as a global health emergency by the World Health Organization(WHO).There are currently no vaccines nor approved drugs for the prevention or treatment of ZIKV infection.Ubiquitin-dependent mitophagy is a mitochondrial quality control mechanism that maintains intracellular environmental homeostasis,through selective degradation of damaged mitochondria.The process includes the initiation of mitophagy,and the formation of autophagy lysosomes mediated by mitophagy receptors.Many viruses promote their infection by either hijacking or antagonizing mitophagy.However,the role of mitophagy in ZIKV infection remains unclear.Therefore,exploring the mechanism of mitophagy in ZIKV replication is helpful to provide new targets for the treatment of ZIKV infection.This study aimed to investigate the role of the mitophagy pathway in ZIKV infection.Firstly,we investigated the effect of ZIKV infection on mitochondria,confirming that ZIKV infection leads to mitochondrial damage.Next,we explored the effects and mechanism of mitophagy activation and the mitophagy receptor-optineurin(OPTN)on ZIKV replication.Methods:1.We reanalyzed the RNA-seq data of cerebral cortex samples of infants with microcephaly caused by ZIKV infection from GEO datasets and screened for differentially expressed genes between ZIKV-infected and uninfected tissues,and further Gene Ontology(GO)pathway analysis was performed to determine the effect of ZIKV infection on mitochondria-related biological processes.Mitochondria were stained with fluorescence in A549 cells,and the effects of ZIKV infection on mitochondrial morphology and functions were observed and analyzed by laser confocal and transmission electron microscopy(TEM).2.Mitophagy was activated by niclosamide(NIC),a mitochondrial uncoupling agent,and inhibited by U0126-EtOH in ZIKV-infected A549 cells.Ubiquitin phosphorylation levels,autophagy activation levels,and ZIKV replication levels were detected to investigate the role of mitophagy activation on ZIKV replication.Furthermore,we constructed a ZIKV-induced microcephaly mouse model by intracranial injection of ZIKV into one-day-old BALB/c mice for in vivo experiments.In this model,the effects of NIC treatment on brain tissue necrosis and ZIKV replication in both blood and brain tissue were detected.Simultaneously,the effects of NIC on mitophagy pathway-related molecules,such as LC3B,PINK1,and Parkin were also detected to verify the role of NIC-induced mitophagy activation in ZIKV replication.To identify the key molecules involved in the inhibitory effect of mitophagy activation on ZIKV replication,PINK1 was knocked down in A549 cells,and its effect on ZIKV replication and NIC-induced antiviral effect was detected at RNA and protein levels.3.The effect of ZIKV infection on the expression level of mitophagy receptor OPTN was detected in both A549 cells and U251-MG cells.We also detected the effect of overexpression or knockdown of OPTN on ZIKV replication,to investigate the role of OPTN in ZIKV replication.To further explore the mechanism of OPTN in ZIKV replication,the effect of OPTN overexpression on type I interferon(IFN)expression and its downstream pathway including phosphorylated STAT1 level,interferon stimulated response element(ISRE)activity,and interferon stimulated genes(ISGs)expression were detected by Western blotting,dual-luciferase reporter assays and Real-time PCR respectively in A549,U251-MG,and 293T cells.Results:1.ZIKV infection affected mitochondria-related biological processes including mitophagy,leading to fragmentation,decreased activity and functional impairment of mitochondria.By reanalysis,a total of 1569 differentially expressed genes(DEGs)were identified from RNA-seq dataset of ZIKV-infected and uninfected cerebral cortex samples,including 1326 up-regulated genes and 243 down-regulated genes.Multiple DEGs were enriched in mitochondria-related biological processes including mitophagy.Laser confocal and TEM observations revealed that mitochondria were fragmented,less active,and functionally impaired in ZIKV-infected cells compared to uninfected cells.2.Mitophagy inhibited ZIKV replication in vitro.In vitro experiments showed that NIC activated ubiquitin-dependent mitophagy and inhibited ZIKV replication in a dose-dependent manner;U0126-EtOH inhibited mitophagy and promoted ZIKV replication,rescuing the inhibitory effect of NIC on ZIKV replication,suggesting that NIC-induced mitophagy suppressed ZIKV replication in vitro.3.Mitophagy inhibited ZIKV replication in vivo.Data from ZIKV-infected mouse model showed that NIC treatment inhibited ZIKV replication and alleviated microcephaly in vivo,and this inhibitory effect was mediated by mitophagy activation as shown by the translocation of PINK1 and Parkin to the outer mitochondrial membrane,restored ZIKV-induced inhibition of PINK1 autophosphorylation and increased ubiquitin phosphorylation level at the Ser65 in mouse brain tissue.4.PINK1 was the key target for the mitophagy-mediated inhibition of ZIKV.Knockdown of PINK1 promoted ZIKV replication and reversed the anti-ZIKV replication effect of NIC suggesting PINK1 is a key target of mitophagy to inhibit ZIKV replication.5.ZIKV infection increased mitophagy receptor OPTN expression leading to the activation of JAK/STAT signaling to inhibit ZIKV replication.Overexpression of OPTN inhibited ZIKV replication at both RNA and protein levels,and promoted the anti-ZIKV effect of IFN-α.Overexpression of OPTN stimulated the expression of IFN-α and IFN-β,which activated the JAK/STAT signaling pathway as shown by increased STAT1 phosphorylation level and enhanced ISRE activity.This,in turn,stimulated expression of ISGs to exert the inhibitory effect on ZIKV replication.Conclusion1.ZIKV infection affects mitochondrial dynamics leading to fragmentation,decreased activity and functional impairment of mitochondria.2.Mitophagy inhibits ZIKV replication and PINK1 is a key target for the inhibitory effect.3.ZIKV infection increases the expression level of mitophagy receptor OPTN leading to the activation of JAK/STAT signaling to initiate the host anti-ZIKV mechanism. |
