DUT-M Positively Regulates RLR Mediated Antiviral Signaling Pathway By Targeting VISA

For a long time,in the research of cellular antiviral innate immunity,the recognition of viral infection by host has been a hot topic.PAMPs(pathogen associated molecular patterns)of pathogenic microorganisms are recognized by PRRs(pattern recognition receptors)of host can be recognized and activated to induce antiviral immunity.Fundamentally different viral nucleic acids can be recognized by a variety of PRRs such as RIG-?-like receptors(RLRs Among them,RIG-? was activated after recognizing intracellular viral RNA,and the activated RIG-? was recruited into the mitochondrial adaptor protein VISA(also known as MAVS,CARDIF and IPS-1).VISA then acts as a central signaling platform connecting RIG-? and downstream signaling components(such as TRAF2,3,5 and 6,TBK1 and IKK),thereby activating the kinases TBK1 and IKK.These activated kinases further phosphorylate transcription factors IRF3/7 and NF-?B,and the activated transcription factors IRF3/7 and NF-?B enter the nucleus,resulting in the production of downstream antiviral signal proteins(such as interferon type I,proinflammatory cytokines and chemokines).In recent years,the discovery and research of RIG-? adaptor protein VISA has greatly enriched our understanding of antiviral innate immune signal transduction.In order to further explore the antiviral innate immune signal transduction mechanism of RLRs,we used RIG-? as bait to carry out yeast two hybrid screening in order to find more antiviral regulatory molecules associated with RIG-?.Deoxyuridine 5 '-triphosphate nucleoside hydrolase(d UTPase)is a protein that interacts with RIG-?.DUT encodes a homotrimeric enzyme with two subtypes,which can be divided into two subtypes: nuclear isoform DUT(DUT-N)and mitochondrial isoform DUT(DUT-M).Using immunoprecipitation,dual luciferase reporter gene,native PAGE and RT-PCR,we found that DUT-M could interact with RIG-?;Moreover,overexpression of DUT-M promoted the dimerization of IRF3 and the phosphorylation of p65 after infection with Sendai virus(SeV);In addition,overexpression of DUT-M can effectively enhance IFN-? The activation of promoter and the m RNA transcription level of downstream antiviral genes(such as IFNB1,CXCL10 and ISG56);However,nuclear localization of DUT-N had no similar effect.Down regulation and knockout of DUT-M expression can inhibit SeV induced IFN-? activation of promoter and transcription of downstream antiviral gene m RNA.Immunoprecipitation assay revealed that DUT-M interacted with VISA and RIG-? to promote the assembly of VISA-TRAF2 complex and enhance the multi-ubiquitination of TRAF2.In conclusion,DUT-M is a positive regulator of RNA virus innate immune response mediated by RIG-?-VISA.DUT-M interacts with VISA and RIG-? to promote the assembly of VISA-TRAF2 complex,enhance the multi ubiquitination of TRAF2,enhance the dimerization and phosphorylation of IRF3 of p65,and enhance the innate immune response mediated by VISA.