Function And Molecular Mechanism Of PRMT1 In Innate Antiviral Immunity

Innate immunity provides the first line of defense against invading pathogens.Activation of innate immunity requires the recognition of pathogen-associated molecular patterns(PAMPs)through pattern-recognition receptors(PRRs).PRRs recognize invading microbes and initiate a series of downstream signaling events that eventually lead to the production of type Ⅰ interferon(IFN)and proinflammtory cytokines.TBK1 is an essential kinase for the innate immune response against viral infection.However,the key molecular mechanisms regulating the activation of TBK1 remain elusive.Protein post-translational modifications such as phosphorylation,ubiquitination,acetylation,play essential roles in regulating antiviral immunity.Protein arginine methylation is an abundant post-translational modification in eukaryotes and the reaction is catalyzed by protein arginine methyltransferases(PRMTs).Accumulating results confirmed that PRMTs play essential roles in antiviral immune response.PRMT1 is the most predominant type Ⅰ PRMT in mammalian cells,accounts for 90%of cellular PRMT activity.However,the function of PRMT1 in antiviral immune response is almost unclear.In this study,we found that knockdown of PRMT1 significantly attenuated the expression of Ifnb1.Further,we isolated the primary peritoneal macrophages from Prmt1fl/fl and Prmt1fl/fl Lyz2-Cre mice and stimulated them with LPS,poly(I:C),HSV60 or infected them with SeV or herpes simplex virus type 1(HSV-1).Compared to macrophages from-Prmtafl/fl mice,macrophages from Prmta1fl/fl Lyz2-Cre mice exhibited a lower expression of Ifnb1 as well as the secretion of IFN-β after all stimulations and infections.IFN-β plays a critical role in host immune responses against viral infection.We found the replication of VSV was substantially increased in Prmt1fl/fl Lyz2-Cre macrophages than Prmt1fl/fl counterparts upon VSV infection.Akin to VSV infection,we found that the copy number of HSV-1 genomic DNA and viral titers of HSV-1 were also higher in macrophages from Prmt1fl/fl Lyz2-Cre mice than those from Prmt1fl/fl mice upon HSV-1 infection.Moreover,Prmt1fl/fl Lyz2-Cre mice showed higher mortality than Prmt1fl/fl mice upon infection with both VSV and HSV-1.IFN-β protein level in the serum after infection with VSV or HSV-1 was much lower in Prmt1fl/fl Lyz2-Cre mice than in Prmt1fl/fl mice.Furthermore,VSV titers and replication in the spleen,liver,and lungs were significantly higher in Prmt1fl/fl Lyz2-Cre mice compared with those in Prmt1fl/fl mice.Besides.the copy number of HSV-1 genomic DNA and viral titers were significantly higher in the brains of Prmt1fl/fl Lyz2-Cre mice than in those of Prmt1fl/fl mice.Altogether,these data indicated that PRMT1 served as a positive regulator for antiviral immune responses against both RNA and DNA viruses.We next sought to determine the molecular mechanisms by which PRMT1 enhanced IFN-β signaling and antiviral responses.We found PRMT1 promoted the expression of IFNB1 mRNA and the activation of IFNB1 promoter mediated by TRIF,cGAS+STING,MAVS,or TBK1,but not IRF3-5D.These data suggested that PRMT1 may enhance IFN-β signaling by targeting TBK1.To confirm that PRMT1 targets TBK1,we investigated their interaction.Coimmunoprecipitation assay showed that PRMT1 was associated with TBK1.Notably,the interaction between PRMT1 and TBK1 was enhanced upon viral infection.To delineate the domains of TBK1 that are required for the binding of PRMT1,we purified His-tagged TBK1 truncations expressed in E.coli,and found that PRMT1 interacted with TBK1 kinase domain and ubiquitin-like domain in vitro.To investigate whether the function of PRMT1 in antiviral immunity depended on its methyltransferase activity.We pretreated mouse macrophages with type I protein arginine methyltransferases inhibitor MS023.We found MS023 treatment decreased Ifnbl expression upon SeV infection or HSV-60 stimulation in a dose-dependent manner,and the phosphorylation of TBK1 was reduced upon SeV infection or HSV-60 stimulation after MS023 treatment.Next,we assessed whether PRMT1 could promote the asymmetric arginine methylation of TBK1.We transfected Myc-TBK1 and FlagPRMT1 into HEK293T cells,and we found the methylation of TBK1 was readily detected in the presence of Flag-PRMT1 after SeV infection.Further,we found the methylation of TBK1 was much lower in Prmt1fl/fl Lyz2-Cre macrophages than in Prmt1fl/fl macrophages upon SeV or HSV-1 infection.We performed in vitro methylation assay with recombinant His-PRMT1 and GST-TBK1 proteins.We found TBK1 methylation was increased in the presence of His-PRMT1(WT),but not in the presence of its mutant His-PRMT1(VLD-AAA).In addition,we found that the methylation of TBK1 54A,80A,134A,228A,229A,and 271A was significantly decreased,which inferred that these 6 arginine residues likely are the key-CH3 group accession sites.To investigate the effect of PRMT1 on the activation of TBK1,we performed several experiments.We found the kinase activity of TBK1 was greatly decreased in PrmtIfl/fl Lyz2-Cre macrophages compared to that in PrmtIfl/fl macrophages after infection with SeV or HSV-1.To directly evaluate whether PRMT1 regulates TBK1 activity,we performed in vitro kinase assays with recombinant GST-TBK1,His-IRF3,and GST-PRMT1 or GST-PRMT1(VLD-AAA)proteins.We found the phosphorylation of TBK1 and IRF3 was increased in the presence of GST-PRMT1.To investigate whether the aforementioned arginine residues were involved in PRMT1mediated TBK1 activation,we transfected 6 single-arginine-mutant TBK1 expression plasmids into HEK293T cells.Compared to TBK1(WT),54A,134A,and 228A mutants showed almost abolished TBK1 phosphorylation in the presence or absence of PRMT1.We reintroduced Myc-TBK1(3A)into Tbk1-/-MEFs.Western blot analysis showed that Myc-TBK1(3A)could not restore TBK1 phosphorylation as Myc-TBK1(WT)did.The dimerization and aggregation of TBK1 are essential for its transautophosphorylation.To investigate whether PRMT1 promotes the oligomerization of TBK1,we utilized Native-PAGE analysis and observed that endogenous TBK1 oligomerization was potently increased upon SeV or HSV-1 infection,while this induced aggregation was significantly attenuated after the deletion of PRMT1 in the macrophages.We also showed that PRMT1 accelerated TBK1 oligomerization in vitro with recombinant TBK1 and PRMT1 proteins.To directly verify whether methylation was involved in the proceeding of TBK1 aggregation,we transfected Myc-TBK1(WT)or Myc-TBK1(3A)together with Flag-PRMT1 into Tbk1-/-MEFs.We found that transfection of Flag-PRMT1 promoted TBK1(WT)aggregation,while Myc-TBK1(3A)could not form oligomers even in the presence of Flag-PRMT1.In summary we identify PRMT1,a type I protein arginine methyltransferase,catalyzes asymmetric methylation of R54,R134,and R228 of TBK1.This kind of modification enhances TBK1 oligomerization after viral infection,which subsequently promotes TBK1 phosphorylation and downstream IFN-β production.More importantly,myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA viruses and RNA viruses than Prmt1fl/fl mice.Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.