OTUD5 Regulates The Innate Immunity Against DNA Viruses By Mediating Deubiquitination Of STING

The innate immune system in mammals has developed unique sensing strategies for detecting pathogenic bodies,including a central strategy based on DNA recognition.Cyclic gmp-amp synthase(cGAS)can detect the DNA released by pathogens or damaged cells,stimulate its conformational changes,induce its enzyme activity,and thus produce endogenous second messenger cGAMP.cGAMP binds to endoplasmic reticulum adaptor protein STING to activate STING.The activated STING was transported from the endoplasmic reticulum through the golgi body to the perinuclear region,thereby activating the downstream signaling pathway and initiating the immune response.Although STING is critical for protecting the host against a variety of DNA pathogens,continuous STING activation can lead to a fatal disease response.Therefore,the intensity and duration of STING signaling need to be dynamically regulated in a multi-layered and highly ordered manner to maintain immune homeostasis.STING is a critical adaptor protein for effective antiviral and anti-tumor innate immunity.Its activity is significantly regulated by ubiquitination.Like most modification after translation,ubiquitin is a reversible process,according to the relevant report about 100 different mammalian genome encoding to ubiquitin enzyme(DUB),including ovarian tumor associated protease(OTUs)is a newly discovered to ubiquitin enzyme family,its OTUD5 is one of the very important member of the family.There are reports for OTUD5 can pass to of ubiquitin Traf3 involved in the natural antiviral immunity,but there are also the reported literature OTUD5 may participate in regulating the adaptive immunity.At present,the study on the deubiquitination of STING is not in-depth enough,the reported DUB does not have OTUs family members,and the study on STING by DUB still needs to be improved systematically.Our research focused on screening members of the OTU family,determining their functions and mechanisms,and then exploring their role in the innate immune antiviral pathway.In this paper,we found that OTUD5 played a k48-bit deubiquitination effect on STING,which could stabilize STING and play an antiviral role in the DNA virus signaling pathway.Objectives:To investigate the function and mechanism of OTUD5 in the innate immunity against DNA virus,and to verify the antiviral effect of OTUD5 through mouse animal model experiments.Methods:1.Screen the DUBs from the OTUs family that play a role in de-ubiquitination of STING.DUBs that have deubiquitination effects on STING were screened by exogenous overexpression plasmids and verified by endogenous deubiquitination experiments.The results showed that OTUD5 played a significant deubiquitination effect on STING.2.Explore the interaction of OTUD5 and STING.The interaction between OTUD5 and STING was analyzed by exogenous overexpression immunocoprecipitation,and the interaction between OTUD5 and STING was further verified by endogenous Co-IP experiments.3.Explore the adjustment of OTUD5 to the stability of STING.Transfect Myc-STING,Flag-OTUD5 and wild-type and mutant ubiquitin plasmids into 293T cells,and then detect the ubiquitination of STING.In order to further verify whether OTUD5 can regulate the stability of STING,we took primary peritoneal cells of Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice and infected them with HSV-1 virus to detect the expression of STING4.Investigate the effect of OTUD5 on cytokine expression mediated by innate immune antiviral signaling pathway.4.1.Detection of downstream cytokine expression after knocking down OTUD5.Wild-type mice of appropriate age were used to obtain primary peritoneal macrophages.48 hours after transfection with siRNA,the cells were stimulated with ISD,HSV-60,cGAMP,LPS,and SeV and HSV-1,and total RNA was extracted and reverse transcribed into cDNA.The expression of Ifnbl and IFN-related genes was detected by real-time quantitative PCR,and the IFN-β secretion was detected by ELISA.4.2.Detection of downstream cytokine expression after knocking out OTUD5.Primary peritoneal cells from Otud5 fl/Y and fl/Y Lyz2-Cre mice were stimulated with ISD,HSV-60,cGAMP,LPS,SeV,VSV,and HSV-1.Total RNA was extracted and reverse transcribed into cDNA.The expression of Ifnbl and IFN-related genes was detected by real-time quantitative PCR,and the IFN-β secretion was detected by ELISA.5.Investigate the effect of OTUD5 on signal pathway activation.Wild-type mice of appropriate age were used to obtain primary peritoneal macrophages.48 hours after transfection with siRNA,the cells were stimulated with ISD,HSV-60,cGAMP,LPS,and SeV and HSV-1 Western Blotting was used to detect TBK1,IRF3 phosphorylation level.Primary peritoneal cells from Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were stimulated with ISD,HSV-60,cGAMP,LPS,SeV,VSV,and HSV-1.Western Blotting was used to detect phosphorylation of TBK1 and IRF3 Level.Non-denaturing Western Blotting was used to detect the dimerization of IRF36.Investigate the effect of OTUD5 on virus replication.Primary peritoneal cells of Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were isolated,and the cells were infected with VSV and HSV-1.Real-time quantitative PCR was used to detect changes in Ifnb1 mRNA levels and viral mRNA levels.Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were injected intraperitoneally with HSV-1 to examine the survival rate.At the meanwhile,and the brain tissues of the mice were isolated for the quantification of the number of viral genomic DNA copies and immunohistochemistry.Results:1.OTUD5 has obvious deubiquitination effect on STING.OTUD5 was screened by exogenous overexpression related plasmids to play a significant deubiquitin effect on STING,while other molecules in the OTUs family did not play a deubiquitination effect on STING.After deactivating the OTUD5 enzyme active site,the deubiquitination effect of OTUD5 on STING disappeared.Primary-peritoneal macrophages were obtained from wild-type mice of appropriate age,and 48 hours after transfection of siRNA,HSV-1 infected cells,and endogenous deubiquitination disappeared after interference with OTUD5.Gives the same result2.OTUD5 interacts with STING.Co-immunoprecipitation experiments proved that OTUD5 and STING directly bind.Mutating the OTUD5 enzyme active site did not affect the interaction between OTUD5 and STING.Transfection of OTUD5 with gradients showed that OTUD5 played a stable role in STING.3.OTUD5 promotes the stability of STING.Transfection of Myc-STING,Flag-OTUD5 and wild-type and mutant ubiquitin plasmids into 293T cells,it can be found that OTUD5 plays a role of de-ubiquitination of K48 at STING,through exogenous and intrinsic detection Stable for STING.4.Knockdown of OTUD5 reduces expression of downstream cytokines.C57BL/6 mice of appropriate age were used to obtain primary peritoneal macrophages,and 48 hours after transfection with siRNA,the cells were stimulated with ISD,HSV-60,cGAMP,and HSV-1 was used to infect the cells.Real-time fluorescence quantitative PCR was used to detect downstream cytokine mRNA levels.The results showed that after interference with OTUD5,ISD,HSV-60,cGAMP stimulation,and HSV-1 infection-mediated Ifnb1 mRNA levels decreased significantly,which indicates that OTUD5 positively regulates the IFN-β mediated by DNA signaling pathway produce.5.Knockout of OTUD5 reduces expression of downstream cytokines.Primary peritoneal cells of Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were stimulated with ISD,HSV-60,and cGAMP,and HSV-1 was used to infect the cells.Real-time fluorescence quantitative PCR was used to detect changes in downstream cytokine mRNA levels.The results showed that after Knockout OTUD5,the levels of Ifnb1 mRNA mediated by ISD,HSV-60,cGAMP stimulation,and HSV-1 infection were significantly reduced,and the levels of downstream genes Ccl5 and Cxcl10 mRNA were also significantly reduced,further indicating that OTUD5 is positive.Regulates the production of IFN-β mediated by DNA signaling pathways6.OTUD5 promotes DNA signaling pathway activation.After the DNA signaling pathway is activated,it can promote the phosphorylation of TBK1 molecules.Phosphorylated TBK1 molecules cause phosphorylation of IRF3 molecules,and the phosphorylated IRF3 molecules dimerize into the nucleus and promote the production of IFN-β.Wild-type mice of appropriate age were used to obtain primary peritoneal macrophages.48 hours after transfection with siRNA,the cells were stimulated with ISD,HSV-60,cGAMP,and HSV-1 respectively.Western Blot test results showed that IRF3 and TBK1 were in the protein.Phosphorylation levels have a significant downward trend.Primary peritoneal cells from Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were stimulated with ISD,HSV-60,and cGAMP,respectively.Western Blotting detected that after OTUD5 was knocked out,the phosphorylation levels of TBK1 and IRF3 were significantly reduced,and non-denatured Western Blotting detected the dimerization level of IRF3,and the results showed that the IRF3 dimerization level was significantly reduced after OTUD5 was eliminated7.OTUD5 has antiviral ability in the body.Otud5 fl/Y and Otud5 fl/Y Lyz2-Cre mice were injected intraperitoneally with HSV-1 virus for 72 hours.The brain tissue was ground and the supernatant was obtained.By qPCR technology detection,it was found that the DNA replication ability was increased after OTUD5 was eliminated.Using mouse lethality experiments to test survival rate,we can see that mice died faster after OTUD5 was knocked out.Brain tissue was taken to detect HSV-1 virus replication by immunohistochemistry.The results suggest that HSV-1 was removed after OTUD5 was knocked out.Enhanced virus replication.Conclusion:1.OTUD5 downregulates the K48-linked polyubiquitination of STING and promotes its stability.2.OTUD5 can promote the ability of cells or organisms to resist DNA viruses.