TRIM25 Ubiquitination Modulates The IFN-? Production Through RIG-I Signaling Pathway In Duck

Avian influenza virus(AIV)is an important pathogen endangering the current poultry industry.It not only causes huge economic losses to the poultry industry,but also causes public health and safety incidents.Therefore,the effective support working resistance of AIV has become one of the urgent problems to be solved in the healthy production of poultry industry.Studies has been found that duck showed lower susceptibility to influenza virus than that of chicken.It may be that the specific RIG-I signaling pathway in duck can be activated to exert antiviral effects.In immune signaling pathways,ubiquitination is widely existed,which can regulate cell physiological functions of cells and replication and proliferation of viruses.TRIM25(tripartite motif containing 25)is an E3 ubiquitin ligase,there remains considerable room for advanced exploration due to the mechanism involved in TRIM25 ubiquitination regulating antiviral effects through RIG-I signaling pathway in duck is not completely clear yet.Based on this,in the present study,5'ppp dsRNA was used to mimic viral infection in vivo to infect duck embryonic fibroblasts(DEFs),and ubiquitome combined with co-immunoprecipitation technology was carried out to analyze the regulatory mechanism of duck TRIM25 ubiquitination on RIG-I signaling pathway.The aim of this study was to reveal the molecular mechanism of RIG-I signaling pathway regulating immune response in duck,which can provide a new solution to reveal the mystery of duck specific virus clearance mechanism and the prevention and control of AIV.The research was carried out as follows:1.The effect of duck RIG-I signaling pathway protein ubiquitination during AIV mimics infection in vitroTo investigate the effect of RIG-I signaling pathway protein ubiquitination in ducks during AIV infection,5'ppp dsRNA was used to mimic AIV infection in vitro.The protein level of total ubiquitination was significantly changed during 5'ppp dsRNA stimulation by Western Blot.In addition,the mRNA levels of RLRs(RIG-I,MDA5,LGP2)and immune inflammatory factors(including IFN-?,IFN-?,TNF-? and IL-2)were significantly up-regulated after 5'ppp dsRNA stimulation.In order to screen the key ubiquitination modification proteins during 5'ppp dsRNA infection in DEFs,label free technique combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)were performed.There were 943 differentially expressed ubiquitination proteins(446 up-regulated and 497 down-regulated)and 1566 differentially expressed ubiquitination sites(754 up-regulated and 812 down-regulated).Meanwhile,35 ubiquitinated motif elements were identifed.Furthermore,the subcellular localization,biological function and KEGG pathway analysis showed that the differentially expressed ubiquitinated modified proteins were mainly related to proteasome degradation,protein localization and energy metabolism.Among them,eight significant ubiquitination proteins and seventeen ubiquitination sites involved in RIG-I signaling pathway(including TRIM25?MAVS?USP14?UBE2N?DDX3X?RAD23A?MEX3C?EFTUD2)were activated by 5'ppp dsRNA stimulation.Furthermore,TMT was performed to verify the changes of the above-mentioned eight protein expression during 5'ppp dsRNA stimulation.The results indicated that ubiquitination rather than protein expression played a key role in duck anti-avian influenza virus infection.2.The anti-viral activity of duck TRIM25 ubiquitination mediating RIG-I signaling pathwayIn order to further investigate the anti-viral function of duck TRIM25 ubiquitination,full-length duTRIM25 cDNA(GenBank accession number:KY974316)was obtained,and tissue expression profile of duTRIM25 was detected.The results showed that duTRIM25 included one RING domain,two B-Box domains and one SPRY domain.Tissue expression profile showed that duTRIM25 mRNA level was highly expressed in the kidney,liver and muscle,while lower expressed in the duodenum and ovary.To futher explore whether duck TRIM25 ubiquitination modulates anti-viral activity through RIG-I signaling pathway,TRIM25 eukaryotic expression vector and interfering RNA were constructed to detect the effects of overexpression and knockdown of TRIM25 on the regulation of RIG-I signaling pathway.Co-immunoprecipitation and western blot were performed to demonstrate that TRIM25 could be ubiquitinated by overexpression of TRIM25.And the ubiquitination level of TRIM25 was significantly enhanced after stimulation with 5'ppp dsRNA.Then,luciferase reporter assays and ELISA analysis showed that overexpression of duTRIM25 could significantly facilitate the activation of IRF1 and NF-?B,and induce IFN-P transcription during 5'ppp dsRNA,while knockdown of duTRIM25 showed the opposite trend.The results of RT-qPCR indicated overexpression of duTRIM25 could enhance the mRNA expression of IFN-?,IRF1,NF-?B,RIG-I,MAVS,FADD,TRAF2 and TBK1 in the RIG-I signaling pathway,while knockdown of duTRIM25 could significantly increase the mRNA expression of the above genes.Hence,duTRIM25 ubiquitination can mediate RIG-I signaling pathway to activate anti-viral immune response during 5'ppp dsRNA stimulation.3.The anti-viral mechanism of duTRIM25 ubiquitination on RIG-I signaling pathwayIn order to further elucidate the anti-viral mechanism of duTRIM25 ubiquitination in activating RIG-I signaling pathway,according to the two kinds of ubiquitination:K48-linked polyubiquitination(proteasome degradation)and K63-linked polyubiquitination(DNA damage repair and NF-?B pathway regulation),we first proved that K63-linked polyubiquitination of duTRIM25 was promoted during 5,ppp dsRNA,suggesting that downstream signal transduction could be activated duTRIM25 ubiquitination.To further confirm the activation mechanism of duTRIM25 ubiquitination,site-directed mutation was performed to mutate lysine sites(K263,K276,K281 and K293)of duTRIM25 according to ubiquitination proteomics results and different lysine mutation sites expression vectors were constructed(K263R?K276R?K281R and K293R),respectively.Co-immunoprecipitation combined with Western Blot results showed that the ubiquitination level of duTRIM25 mutants at position 276 and 293 were significantly reduced during 5'ppp dsRNA stimulation.Therefore,the lysine sites of duTRIM25 at position 276 and 293 can activate RIG-I signaling pathway to regulate the antiviral activity.Taken together,duTRIM25 ubiquitination can promote IFN-? expression to mediate the RIG-I signaling pathway and play an important role in anti-viral immune response based on the lysine sites of duTRIM25 at position 276 and 293.These results elucidated the regulatory mechanism of duTRIM25 ubiquitination mediating RIG-I signaling pathway.