ARIH1 Promotes Antiviral Immunity And Autoimmunity By Inducing Mono-ISGylation And Oligomerization Of CGAS

Immunity is a physiological process that maintain a stable internal environment by recognizing self and eliminating non-self.The immune system of higher animals consistes of innate immunity and adaptive immunity.Innate immune systems recognize pathogen-related molecular patterns（PMAPs）and danger-associated molecular patterns（DMAPs）by pattern recognition receptors（PRRs）,then induce series of signal cascade reactions and the expression of inflammatory cytokines to initiate innate immune response,and also promote adaptive immune response.Therefore,innate immunity plays a fundamental role in immune response.Understanding its regulatory mechanisms can help to reveal the molecular mechanisms of infectious and inflammatory diseases,and provide theoretical basis and molecular targets for the treatment of related diseases.CyclicGMP-AMP synthase（cGAS）is an important PRR.cGAS undergo oligomerization after recognizes cytoplasimc DNA,and converts ATP and GTP into cGAMP.cGAMP binds to the downstream adaptor protein MITA（also known as STING）to activate the downstream signaling pathway and induce type I interferons and pro-inflammatory cytokines expression.Knockout of cGAS promotes DNA virus infection and inhibits the inflammatory response caused by self-DNA,indicating its significance in antiviral immunity and autoimmunity.Human immunity can be classified into innate immunity and adaptive immunity.Research from our group and other laboratories has shown that various post-translational modifications regulate the activity and homeostasis of cGAS.For example,RNF185 catalyzes cGAS ubiquitination at K173 and K184 residues,which enhancs the activity of cGAS,thereby thus promoting antiviral innate immune signal transduction.AKT1 mediates cGAS phosphorylation at S305,inhibits the activity of cGAS enzyme,and reduces expression of cGAMP and type I interferon.TRIM38catalyzes sumoylation of cGAG at K217 and K464,inhibits the degradation of cGAS and maintains its stability.USP29 induces deubiquitination of cGAS at K271,maintains the stability of cGAS,and promotes antiviral innate immune signal transduction and autoimmunity.However,further study is needed to determine whether there are additional types of post-translational modification of cGAS,as well as their functions in regulating antiviral immunity and autoimmunity.In this study,we used cGAS as the bait protein to conduct yeast two-hybrid experiments with over 200 human E3 ubiquitin ligase yeast expression vectors previously constructed by our laboratory.Positive clones were constructed into mammalian cell expression vectors and screened with cGAS by immunoprecipitation.An E3 ubiquitin ligase interacting with cGAS,ARIH1 was identified.Endogenous co-immunocoprecipitation experiments confirmed that ARIH1 can interact with cGAS constitutively,and HSV-1 infection can further promoted it.Knockdown of ARIH1 in THP-1 cells inhibited the activation of IRF3 and NF-κB,and expression of type I interferon and other cytokines induced by HSV-1 or cytoplasm DNA.To further investigate the role of ARIH1 in vivo,ARIH1 conditional knockout mice(Arih1^fl/fl)were constructed by CRISPR/Cas9 technology,and then Arih1^fl/flknockout mice were hybridized with Lyz2-Cre or Cre-ER mice to obtain Lyz2-Cre;Arih1^fl/fland Cre-;Arih1^fl/flmice.RT-PCR,Wsternblot and ELISA experiments proved that knockout ARIH1 in bone marine-derived dendritic cells（BMDCS）,macrophages（BMDM）,or lung fibroblasts（MLFS）,significantly inhibited expression of type I interferon and inflammatory cytokines induced by HSV-1 infection or cytoplasmic DNA,and promoted HSV-1 replication.These results suggested that ARIH1positively regulates HSV-1 infection or cytoplasmic DNA-induced innate immune signal transduction.Compared with control mice,after HSV-1 infection,Lyz2-Cre;Arih1^fl/fland Cre-;Arih1^fl/flmice showed increased mortality,suppressed expression of type I interferon and pro-inflammatory factors in multiple organs,and lower serum protein levels of type I interferon and pro-inflammatory factors.TREX1is cytoplasmic DNA-degrading exonucrease and its inactivation leads to autoimmune diseases like Aicardi-Goutières syndrome（AGS）.Trex1^-/-mice exhibite severe systemic inflammatory responses and lethal autoimmune phenotype.This study revealsed that knocking out of ARIH1 in myeloid cells inhibited systemic inflammatory responses in Trex1^-/-mice and completely rescued their lethal autoimmune phenotype.These findings suggested that ARIH1 positively regulates anti-DNA virus immune responses and cytoplasmic DNA-induced autoimmunity.This study further investigated the molecular mechanism of ARIH1 regulating antiviral immunity and autoimmunity.First,we found that reconstituted with wide type ARIH1,but not its inactive mutation ARIH1^C357S,restored the expression of cytokines such as type I interferon induced by HSV-1 infection or cytoplasmic DNA in ARIH1 knockout cells,suggesting that ARIH1 positively regulates cytoplasmic DNA-induced innate immune responses dependent on its E3 ubiquitin ligase activity.Secondly,knocking out of ARIH1 inhibited cGAS oligomerization and downstream cGAMP production induced by HSV-1 infection or cytoplasmic DNA-induced,but not affected the expression of downstream genes such as type I interferon induced by cGAMP,suggesting that ARIH1 primarily functions at cGAS level.Thirdly,this study confirmed that ARIH1 induced mono-ISGylation of cGAS via cell and biochemical experiments,and this modification promoted DNA-induced oligomerization of cGAS.Finally,mass spectrometry experiments identified the K187residue of cGAS as the modification site.The mutation of this site enhanced DNA-induced oligomerization of cGAS,suggesting that K187 is the site for the inhibition of cGAS activity,and ARIH1-mediated mono-ISGylation of this site relieved the inhibition of cGAS activity,promoted the oligomerization activation of cGAS.In summary,our study revealed that ARIH1 promotes DNA-induced oligoactivation of cGAS by mediating mono-ISGylation of K187 on cGAS,thereby positively regulating antiviral immunity and autoimmunity.Our study provided molecular targets and theoretical basis for treatment of viral infections and autoimmune diseases.