The Role And Mechanism Study Of OTUD6B In Antiviral Innate Immunity

Background:In the process of viral invasion,the innate immune system of the body as the first line of host defense mainly recognizes viral nucleic acids through Pattern Recognition Receptors(PRR).Different viral nucleic acids can be recognized by the host corresponding receptors,including RIG-Ⅰ like receptor(RLR),Toll like receptor(TLR)and cytoplasmic dsDNA sensor cGAS.For example,RIG-Ⅰ like receptors can induce the aggregation of mitochondria)adaptor protein MAVS.The activation of the DNA receptor cGAS can induce the production of the second messenger cGAMP,which in turn induces the dimerization of STING and its translocation from the endoplasmic reticulum to the Golgi apparatus.Then,the downstream TBK1 is recruited and autophosphorylated,which leads to the activation IRF3 and NF-κB transcription factors.The nucleation of IRF3 finally induces the production of IFN-Ⅰ and the subsequent a serious of antiviral immune responses.As an important regulator of IFN-β transcription,IRF3 is activated by upstream TBK1/IKKεthrough phosphorylation and enters the nucleus in the form of the dimer.By interacting with CBP/P300 coactivator,IRF3 binds to the upstream cis-acting elements of IFN-βand enhances IFN-β transcription,which takes an important role in the host defense against viral invasion.However,excessive IRF3 response can also lead to many diseases,such as fat inflammation,insulin tolerance,and systemic lupus erythematosus.Therefore,it is vital to explore the mechanism of IRF3 regulating the innate antiviral immune response.Ubiquitination as the post-translational modification of cellular proteins can regulate many cellular processes,including cell division,cell autophagy,inflammation,apoptosis,etc.The deubiquitinase(DUB)can specifically bind to its target protein,remove ubiquitin and dissociate the polyubiquitin chain,which is a reverse process of ubiquitination..Among the DUB family,all members of the Ovarian tumor domain(OTU)deubiquitinase subfamily were named for having OTU domain or similar structure domain.Most of the members of this subfamily have been reported in mediating cell signaling cascade.Because of the significant changes in structure and function,members of the OTU subfamily show different regulatory mechanism.At present,the role and mechanism of the OTU family regarding its antiviral function are not clear,and the relative research is also few.To fully understand the role and mechanism of the OTU family in antiviral immune response,we constructed the CRISPR/Cas9 knock down library of the OTU subfamily.Using VSV virus as the tool virus,we identified that OTUD6B has the most significant effect on VSV replication.OTUD6B is a member of the OTU subfamily.It has been reported that it can bind to the protein synthesis initiation complex and t modify the pre-initiation complex.It was also reported to affect protein synthesis in non-small cell lung cancer cells by regulating the downstream related molecules of mTORC1.Besides,OTUD6B can inhibit DNA synthesis and modify different targets to regulate cell growth and proliferation.Recently,it has been reported that OTUD6B may play a role in the assembly and function of proteasomes.However,The function and mechanism of OTUD6B in antiviral innate immune have not been addressed.Methods:(a)The CRISPR-Cas9 Library of OTU subfamily was constructed and stable knock down cell lines were generated.The antiviral effect of individual OTU subfamily members was screened by VSV infection.(B)The up-regulation or downregulation of OTUD6B was performed to verify its role in regulating virus infection.(C)We detected the relationship between OTUD6B and IFN-β by dual luciferase reporter assay,Real-time PCR and ELISA.We identified the OTUD6B target protein acting in IFN-β signaling pathway.(D)The correlation between OTUD6B and IRF3 was detected by Western blot and immunofluorescence.(E)Different types of ubiquitination plasmids,such as HA-UB-K6 and HA-UB-K11,were cotransfected with Flag-IRF3 and OTUD6B overexpression plasmids or down-regulation plasmids.The specific type of deubiquitination of IRF3 regulated by OTUD6B was explored by immunoprecipitation experiments.(F)The specific amino acid sites where OTUD6B worked on IRF3 were determined by constructing different ubiquitination deficient mutants of IRF3,Results:1.OTUD6B inhibits the replication of VSV,RSV and SeV.2.OTUD6B promotes the production of IFN-β.3.OTUD6B interacts with IRF3 and promotes IRF3 protein stability.4.OTUD6B reduces the K33 ubiquitination of IRF3.5.OTUD6B reduces K33 ubiquitin of IRF3 ligation at lysine 315.Conclusion:We found that OTUD6B can play a broad-spectrum antiviral function through the deubiquitination of IRF3 K33 modification,which stabilizes the IRF3 stability,promotes the IFN-β production and ISG genes expression downstream of interferon.These findings are expected to provide new therapeutic targets and ideas for the clinical treatment of viral infection.