The Role Of PRMT6 In Antiviral Innate Immunity And The Underlying Mechanisms

Innate immune system can sense and eliminate the invading pathogens,thereby protecting host from infections and damages.The innate immune system recognizes pathogen-associated molecular patterns?PAMPs?of the microorganisms through pattern recognition receptors?PRRs?,initiates innate immune responses,and produces type?interferon?IFN-??and pro-inflammatory cytokines for the defense against infections.RIG-I-like receptors?RLRs?are key receptors in PRRs and play a crucial role in mediating antiviral innate immune responses.RIG-I,the most critical receptor in RLRs,resists viral infection by recognizing the RNA virus and activating TBK1-IRF3 signal through adaptor protein MAVS to initiate IFN-?production.Phosphorylation and ubiquitination and related enzymes play important roles in RIG-I signal initiation,transmission and termination,thus maintaining the immune homeostasis.Based on our previous studies on RIG-I in antiviral immune response,we identified the interacting molecules and post-translational modifications of RIG-I by IP-MS.And we found that RIG-I interacting molecules including methyltransferase,acetyltransferase,E3 ubiquitin ligase,kinase/phosphatase,metabolism and RNA modification related molecules,etc.The types of post-translational modification of mRIG-I include methylation?lysine and arginine?,acetylation,ubiquitination,phosphorylation?serine,threonine and tyrosine?,etc.The function of most interacting molecules and post-translational modifications in RIG-I signal is unknown.Next,we validated the mass spectrometry data and cloned the mutant variants of the modified sites in mRIG-I,aiming to study their functions in the regulation of antiviral innate immune response.Protein arginine methyltransferases?PRMTs?,including 9 family members and mediating methylation of the arginine residues of substrate proteins,are ubiquitously expressed in diverse tissues and cells,which govern the important cellular processes that affect gene expression,pre-mRNA splicing,DNA damage signaling,mRNA translation,cell signaling,and cell fate decision.Althrough the function of PRMTs are widely studied owing to the development of antibody preparation and proteomics techniques,the function of PRMTs in innate immunity,especialy antiviral innate immunity,remains unclear.Among the interacting molecules of RIG-I indentified via mass spectrometry,we found there are several PRMTs molecules,indicating the possible roles of PRMTs in innate immunity.We cloned 9 PRMTs and conducted a functional screening experiment by gene reporter assay.We found that PRMTs displayed different roles in regulating type I interferon expression.Among them,PRMT6 most potently inhibited the expression of IFN-?luciferase reporter induced by RIG-I-2CARD,MAVS,TBK1 and IRF3-5D.We also generated Raw264.7 cells stably overexpressed PRMTs and found that PRMT6overexpression significantly inhibited VSV infection-induced IFN-?production,as well as IRF3 phosphorylation.In addition,we obtained similar results in VSV-infected Raw264.7 cells,NIH 3T3 cells and human A549 cells,in which PRMT6 was transiently overexpressed.These results demonstrated that PRMT6 negatively regulated type I interferon expression in antiviral innate immune responses.In order to investigate the function of PRMT6 in host antiviral response in vivo,we generated the PRMT6-defecient(Prmt6^-/-)mice using CRISPR-Cas9 system.Prmt6defeciency didn't affect the development of immune system in mice.We challenged littermate mice with VSV and found that Prmt6 defeciency significantly increased the survival rate.ELISA analysis of the cytokines in blood showed that Prmt6^-/-mice produced significantly higher levels of IFN-?,IFN-?and IL-6 than Prmt6^+/+mice in response to VSV infection.In consistant with the cytokine production,the replication and viral load of VSV virus in liver,spleen and lung of Prmt6^-/-mice were obviously lower than those from Prmt6^+/+mice.In addition,we observed less infiltration of inflammatory cells into the lung of Prmt6^-/-mice than Prmt6^+/+mice following infection.Altogether,these results confirmed that PRMT6 deficiency enhanced antiviral innate immune response in vivo,validating the function of PRMT6 to inhibit antiviral innate immunity.We further observed that PRMT6 deficiency promoted the production of IFN-?,IFN-?and IL-6,as well as IRF3 activation in bone marrow-derived macrophages?BMDMs?upon VSV or HSV-1 infection in vitro.Nevertheless,the phosphorylation of TBK1 and the activation of mitogen-activated protein kinases?MAPKs?and NF-?B signaling was not affected by PRMT6 deficiency.These results indicated that PRMT6 attenuated antiviral innate immunity by inhibiting IRF3 activation and IFN-?production.Viral infections usually result in the expression change of targeted genes,especially those regulating antiviral innate immunity.By immunoblot analysis of virus-infected cells,we found that viral infections markedly increased PRMT6 expression in mouse and human macrophages.Viral infection also greatly increased PRMT6 expression in human A549 and HepG2.2.15 cancer cells.Meanwhile,the public data from GDS5093,GDS1667 and GDS2164 also indicated that viral infection would up-reguate the expression of PRMT6 and this change may be closely related to the course of the diseases.We further analyzed the cellular localization of PRMT6 and found that PRMT6mainly localized in the cytoplasm in mouse and human macrophages.These results demonstrated PRMT6 as a viral infection-inducible protein,which inhibited IRF3activation in cytosol.The methyltransferase activity was important for the physiological function of PRMTs family members.So,we tried to ascertain whether the function of PRMT6 in antiviral response depended on its methyltransferase activity.We cloned the catalytic inactive form of PRMT6?PRMT6?dead??according to the published reports and found that both PRMT6 and PRMT6?dead?overexpression significantly inhibited the activity of IFN-?luciferase reporters induced by TRIF,STING,RIG-I-2CARD,MAVS,TBK1,IRF3-5D,indicating that PRMT6 exerted its functioin independent on its methyltransferase activity.It is known that TBK1-IRF3 signaling cascade is the common pathway activated by adaptor proteins TRIF,STING and MAVS.Interestingly,our gene reporter assay results revealed that the IFN-?expression induced by adaptor proteins TRIF,STING and MAVS was both siginificantly inhibited by PRMT6,indicating that PRMT6 may target TBK1-IRF3 signal complex.We further demonstrated this proposal by co-transfecting TBK1,IRF3 and PRMT6 in another gene reporter assay.Together with the above results,these results demonstrated that PRMT6 deficiency enhanced the activation of IRF3 without affecting the phosphorylation of TBK1,suggested that PRMT6 may not affect the kinase activity of TBK1.We further demonstrated that PRMT6 didn't affect the kinase activity of TBK1 by kinase assay.Altogether,these results demonstrated that PRMT6 targeted TBK1-IRF3 signal complex,without affecting the kinase activity of TBK1,to inhibit IRF3 actvation in a methyltransferase-independent manner.Since PRMT6 functions independently of its enzymatic activity and does not affect the phosphorylation and kinase activity of TBK1,we wondered whether PRMT6 directly bound to TBK1 or IRF3 to inhibit signal transmission.Co-immunoprecipitation and immunoblot analysis revealed that PRMT6 interacted with IRF3,but not TBK1,IKK?,IRF7.We also examined the interaction of endogenously expressed PRMT6 and IRF3under physiological conditions and found that PRMT6 constitutively interacted with IRF3 in unstimulated THP-1 and BMDMs cells and the interaction between PRMT6 and IRF3 was enhanced in both cell types upon VSV infection.Meanwhile,we observed enhanced TBK1-IRF3 interaction and consequent increased phosphorylation of IRF3 in Prmt6^-/-BMDMs upon VSV infection.We also found that the binding of PRMT6 to IRF3would block further interaction between TBK1 and IRF3,subsequently leading to the inhibition of IRF3 activation and decreased IFN-?production.Altogether,these results suggest that PRMT6 binds and sequesters IRF3,blocks the TBK1-IRF3 interaction and inhibits the activation of IRF3 and subsequent IFN-?production upon viral infection.To further determine which domain of PRMT6 is responsible for its function,we constructed PRMT6 mutants with deletion of certain domains according to its domain structure.We found that the N-terminal domain of PRMT6 was sufficient for the interaction with IRF3.Additionally,co-immunoprecipitation and immunoblot analysis showed that PRMT6 blocked the TBK1-IRF3 interaction and subsequent IRF3phosphorylation,but PRMT6??189-318?rescued these effects while other mutants of PRMT6 failed.Collectively,these data indicated that the AA189-318 domain of PRMT6was critical for blocking the TBK1-IRF3 interaction and inhibiting IFN-?production.These results also indicated that PRMT6 inhibited IRF3 activation and antiviral innate immunity by binding to IRF3 through its N terminal domain and subsequently blocking TBK1-IRF3 assembly dependent on its AA189-318 domain through steric effect.Taken together,in this study,we have identified the interacting molecules and post-translational modifications of RIG-I through IP-MS and revealed the negative regulatory role and underlying mechanisms of PRMT6 in antiviral innate immunity.Our results correlate PRMTs with innate immunity and provide new insights for studying the function of PRMTs and also suggest a possible target for the prevention and treatment of viral infectious diseases.Meanwhile,the detailed mechanisms revealed in our study may represent a new immune evasion strategy for virus,which depeens our understanding of the interaction of virus and host.