YTHDF3 Negatively Regulates Type I Interferon-mediated Innate Antiviral Responses And The Underlying Mechanism

The innate immune system,selected by evolution,is the first line of defense against invading viruses.Type I interferons(IFN-I),namely interferon-a(IFN-a)and IFN-?,are vertebrate-dervied and protective in acute viral infections,which initiate a signaling cascade through the Janus kinase signal transducer and activiator of transcription(JAK-STAT)pathway,leading to transcription of thousands of IFN-stimulated genes(ISGs).Host manipulates these ISGs to restrain different steps of viral life cycle.The dysregulation of IFN-I antiviral responses would cause impairement of antiviral immunity or pathoegensis of inflammatory autoimmune diseases.Therefore the homeostasis of type I interferons-mediated antiviral effector responses is pivotal for host health,which is tightly regulated.The basal IFN-I signaling pathway is maintained homeostatically at low levels by an autocrine loop,and innate immue cells can respond rapidly to low levels of IFN-I because of the pre-existing repertoire of constitutive host defence factors.These.The synthesis of these antiviral immune effectors and regulators is accomplished at transcriptional,translational and post-translational levels.Translational control favors the cell to adjust itself timely adaptation to maximize survival under stress by regulating the translation rate of select mRNAs,especially ideal to host initiation of antiviral innate immune responses.Through utilizing the cellular translation machinery,the host could produce more antiviral effectors.Therefore,selective translational regulation of gene expression is vital to the control of protein abundance,which determines the innate antiviral responses.However,the translation control regulators of innate antiviral responses remain to be further identified.It's well accepted that the epigenetic modifiers are involved in almost all the physiological and pathological processes.How the epigenetic modifiers determine and regulate the immune cell development and functions,and what roles they paly in the immune homeostasis and even the pathogenesis of immune disorders are the hot topics in the field of immunology.However,so many unanswered question remain to be addressed.Here we report that the m6A reader YTHDF3 negatively regulates IFN-I-mediated antiviral innate responses by promoting translation of the repressive transcription factor of innate antiviral response,forkhead box protein O3(FOXO3).To investigate whether the m6A readers YTHDF1,YTHDF2 and YTHDF3 are involved in the antiviral innate responses,we ultilized siRNA interfering or CRISPR-Cas9-knocking out YTHDF1,YTHDF2 and YTHDF3 in macrophages and then found that only knockdown or knockout of YTHDF3 significantly inhibited VSV replication.Furthermore,YTHDF3-deficient cells were also resistant to EMCV and HSV-1 infection,and on the other hand,these viruses inhibited YTHDF3 expression.These results indicated that YTHDF3 might be involved in the interaction of virus and host in a feedback way.Intriguingly,the deficiency of YTHDF3 induced the upregulation ISGs,but YTHDF3-/-Ifnar I-/-macrophages showed no significant difference of ISGs and VSV replication,so we demonstrated that YTHDF3-mediated negative regulation of ISGs expression was dependent on IFNAR1 signaling.YTHDF3-/-mice were protected from VSV infection in vivo.To determine the molecular mechanism of antiviral resitance in YTHDF3-deficient cells,we performed an enhanced CLIP-seq assay,and found that YTHDF3 binded translation initiation region of FOXO3 mRNA.Deficiency of YTHDF3 inhibited translation of FOXO3.Only full-length YTHDF3 protein could bind its target RNAs.We generated mutants,YTH domain,deletion of YTH domain and Pro/Gln/Asn-rich domain,and four residue mutants,which K422 and R533 on the surface of YTH are involved in binding to the RNA backbone,and W438 and W492 within the hydrophobic pocket specifically recognised m6A RNA.We found that all these mutants abrogated its binding of target mRNAs.To investigate the molecular fuction of YTHDF3,we utilized co-IP with MS analysis of YTHDF3 protein interactome,and identified that YTHDF3 interacted with translation initiation factors and kinds of regulatory RNA binding proteins(RBPs),which indicated that YTHDF3 regulated antiviral immune effectors expression at the translational control level.We found that YTHDF3 could coopretate with PABP1 and eIF4G2 to promote translation of FOXO3.Through rescuing FOXO3 expression in YTHDF-/-MEFs,we demonstrated that FOXO3 was definitely pivotal to YTHDF3-mediated negative regulation of innate antiviral immunityOur study demonstrates that YTHDF3 negatively regulates IFN-I-mediated antiviral innate responses through promoting FOXO3 protein expression.Theses findings establish a previously unknown translation-control model in the regulation of antiviral innate responses.Furthermore,as a physiological feedback regulator,YTHDF3 might not only serve as a potential therapeutic target to boost basal ISGs expression,but also be involved in the pathogenesis of tumors and autoimmune diseases.