The Role And The Underlying Mechanism Of Exosomes In Zika Virus Infection

BackgroundZika virus(ZIKV)belongs to the Flaviviridae family with a positive,single-strand ribonucleic acid(RNA).Zika virus infection causes a public health concern because of its potential association with the development of microcephaly.Currently,no approved vaccines or therapies are available for the prevention and treatment of ZIKV infection,therefore it is essential to study the pathogenesis of ZIKV.In the battle between pathogens and host,Interferons(IFNs)play an important role in regulating the host immune response to fight many pathogens.Furthermore,IFN?responds in the early stage of host defense.By binding to a ubiquitously expressed heterodimeric receptor that is composed of the IFN-? receptor 1(IFNAR1)and IFN-?receptor 2(IFNAR2)subunits,type ? IFNs(maily IFN-? and IFN-?)activate the Janus kinase/signal transducer and activator of transcriptions(JAK/STAT)signaling pathway to induce the expression of several hundreds of interferon-stimulated genes(ISGs).In addition,IFN? can also evoke immunomodulatory,antiviral,anti-tumor,and anti-inflammatory responses through induced expression of several genes to activate NK cells.Previous research demontrated that exosomes function as crucial regulators of cellular cross-talk.Exosomes are phospholipid bilayer-bound structures that can transfer packed RNAs,proteins and lipids into recipient cells to facilitate intercellular communications and to affect gene expression in recipient cells.Emerging evidence indicated that exosomes can mediate the transfer of pathogen-derived antigens and virulence factors.In addition,in some cases exosomes participate in the transmission of anti-viral cargo from these sites to other(s)within human body.Here,we aim to clarify whether ZIKV-induced exosomes can regulate viral pathogenic by transferring specific RNAs.Furthermore,we also focus on clarify whether IFN?induced exosomes can regulate the cytotoxicity of NK cells by transferring specific lncRNAs into NK cells.MethodsIn order to further decipher the role of exosomes in the pathogenesis of ZIKV infection,we used human transcriptome array(HTA)and bioinformatics analysis to identify candidate molecules in ZIKV-induced exosomes.Quantitative Real-time PCR(qPCR),western blotting,ELISA,CCK-8,and flow cytometry were used to characterize the mRNA and protein expression levels,cell proliferation and cell cycle progression,respectively.In the second part of study,exosomes were isolated from the supertanants of A549 cells with or without IFN? treatment.Co-culture and ELISA assay were used to analyze the effect of exosomes on the cytotoxicty of NK cells.Human transcriptome array(HTA)was performed to analyze the expression pattern of RNAs wrapped in exosomes.Then subcellular location,qPCR,western blotting,dual-luciferase reporter assay and ELISA were used to characterize long noncoding RNAs(lcnRNAs)location,sponge absorb effects,the expression of NKp46 and cytotoxicity of NK cells.Results1.Through human transcriptome array(HTA)we found the expression level of defensin alpha 1B(DEFA1B)was significantly increased within exosomes isolated from ZIKV-infected A549 cells.Additionally,we found that the extracellular DEFA1B but not the intracellular DEFA1B exerts anti-ZIKV activity,mainly before ZIKV entering host cells.Interestingly,up-regulated DEFA1B retards the cell cycle progression of host cells.Further studies demonstrated that DEFA1B interacted with the origin recognition complex subunit 1(ORC1)which is required to initiate DNA replication during the cell cycle.Machanistically,increased DEFA1B expression decreased the ORC1 level in the cell nuclei to retard the cell cycle progression.Accordingly,DEFA1B-containing exosomes can be internalized by the recipient cells to retard their cell cycles.2.IFN?-induced exosomes can strengthen the cytotoxicity of NK cells.Through HTA we found the expression levels of 69 lncRNAs were significantly altered within exosomes isolated from A549 cells following IFN? treatment.Additionally we found a specific exosomal cargo,linc-EPHA6-1,acted as a competing endogenous RNA(ceRNA)for hsa-miR-4485-5p,which subsequently up-regulate the expression level of natural cytotoxicity receptor(NKp46)on NK cells.Furthermore,we verified over-expression of linc-EPHA6-1 significantly enhance the cytotoxicity of NK cells against A549 cells with or without Zika virus infection.ConclusionIn summary,our results demonstrated that the anti-ZIKV activity of DEFA1B can be mediated by exosomes,and DEFA1B interacts with ORC1 to retard cell cycles,which may play an important role in the development of microcephaly.Our study provides a novel concept that DEFA1B not only acts as an anti-viral molecule during ZIKV infection but also may correlate with neurodevelopment by retarding the progression of cell cycles.In the second part,our results demonstrated that IFN?-induced linc-EPHA6-1 wrapped in exosomes can strengthen the cytotoxicity of NK cells.Our study provides a novel link between type ? IFN and NK cells,which are two major players for the host innate immunity against many pathogen infections.