Screening The Key Molecule SNX9 Based On TMT Protein Quantification And Tandem Mass Spectrometry And The Effect Mechanism Of FAdV-4 Infection Immunity Regulating CGAS-STING Pathway

Fowl adenovirus serotype 4(FAd V-4)is a double-stranded DNA capsidless virus belonging to the genus Aviadenovirus of the Adenoviridea family,with the main clinical feature of hepatitis-pericardial hydrops syndrome(HHS).The disease is susceptible to broilers from 3 to 6 weeks of age and causes a high mortality rate of 80%.Also FAd V-4 can be transmitted between ducks and geese,indirectly demonstrating that FAd V-4 infection poses a potential risk to the entire poultry industry.Due to the strong transmission,high pathogenicity and cross-species nature of FAd V-4,HHS has now spread worldwide causing huge economic losses to the poultry industry.SNXs family molecules play an important role in viral replication and in the regulation of natural immunity.However,the relevance of SNXs family molecules in the immune regulation of FAd V-4 infection and the mechanism of action have not been reported.c GAS-STING pathway mediates the signaling pathway of IFN-β production to resist DNA virus infection.ch STING is a membrane protein molecule localized on the endoplasmic reticulum that is finely regulated in both directions,but which molecules regulate this complex process? Can the SNXs family of molecules be involved in the activation of the c GAS-STING pathway by FAd V-4 infection? It has not been studied yet.Therefore,in this study,we investigated the role of SNXs family molecules in the regulation of c GAS-STING pathway by FAd V-4 infection,and the main findings are as follows.1.Quantitative expression profile of TMT proteome in FAd V-4-infected LMH cellsTo investigate the natural immune pathways activated during FAd V-4 infection and changes in SNX9 expression,this study selected 12 h and 24 h time points after FAd V-4 infection of LMH cells and uninfected cells for TMT quantitative proteomics analysis.The results showed that a total of 479,648 spectra,67,277 identified peptides and 16728 proteins were generated.In group B1/A,520 significant DEPs were identified,including 171 up-regulated DEPs and 349 down-regulated DEPs.in group B2/A,a total of 1257 significant DEPs were identified,including 425 up-regulated DEPs and 832 down-regulated DEPs.subsequently,GO,KEGG,COG,Pfam and subcellular localization were used to differentially GO and KEGG enrichment analyses revealed that the pathways associated with differential protein expression in host cells were all involved in the cytoplasmic DNA-sensing pathway of the innate immune system,possibly including the c GAS-STING pathway.The reliability of the proteomic data was also verified by RT-q PCR,which revealed significant upregulation of PACSIN2,USPL1,ATXN10,ZNF507 and SNX9.This section reveals that the cytoplasmic DNA recognition pathway c GAS-STING may play a crucial regulatory role in the early and middle stages of FAd V-4 infection of LMH cells.2.Tandem mass spectrometry screening of ch STING potential regulatory interacting protein-SNX9 in FAd V-4-infected LMH cellsch STING is a key bridging protein of the c GAS-STING pathway and was screened for key proteins capable of regulating ch STING during FAd V-4 infection.In this study,we screened 272 proteins that may interact with ch STING by immunoprecipitation(IP)combined with tandem mass spectrometry identification.We selected 11 genes with high confidence(HSPA8,G3BP1,DDX17,PRDX1,TBK1,HNRNPA2B1,DDX39 B,HNRNPH3,NPM1,PABPC1,SNX9),cloned them and constructed eukaryotic plasmids for expression to investigate their effects on chicken IFN-β expression,and The interaction with ch STING protein was analyzed by immunoprecipitation(Co-IP)and fluorescence co-localization.The results using dual luciferase showed that these genes were able to regulate STING-mediated IFN-β expression.Seven of these proteins(TBK1,HNRNPA2B1,DDX39 B,HNRNPH3,NPM1,PABPC1,SNX9)were able to interact with ch STING,while only SNX9 and TBK1 were able to co-localize cellularly with STING.This section clearly screens for ch SNX9,a key molecule that potentially regulates ch STING upon FAd V-4 infection.3.Role of ch SNX9 in the induction of IFN-β production by FAd V-4 infection in c GASSTINGTo investigate the role of ch SNX9 in the induction of IFN-β production by FAd V-4 infection in c GAS-STING,this study used the E.coli expression system to express ch SNX9 protein and perform subcellular localization analysis.By performing si RNA knockdown and overexpression of ch SNX9 molecules in LMH cells,the cytokines IFN-β,IL-6,IL-1β and anti-viral protein Mx-1 were determined using RT-q PCR in FAd V-4 infected LMH cells.The replication levels of FAd V-4 were quantified using IFA,Western-Blot and fluorescence.The results showed that ch SNX9 protein showed soluble expression and was localized in the cytoplasm.ch SNX9 negatively regulated the production of IFN-β in LMH cells induced by FAd V-4 infection,and overexpression was found to promote FAd V-4 proliferation in the early stage of virus infection,and knockdown ch SNX9 inhibited its replication.This section elucidates the critical role of ch SNX9 in the regulation of natural immunity during FAd V-4 infection.4.Mechanism of immunomodulatory effects of ch SNX9-mediated c GAS-STING pathway on FAd V-4 infectionTo elucidate the mechanism of ch SNX9 regulation of the c GAS-STING pathway during FAd V-4 infection.In this study,p Ds Red-Monomer-Golgi,p EGFP-MitoDs Red2 and p Ds Red2-ER plasmids and ch SNX9 were cotransfected with LMH cells to further investigate the localization of ch SNX9 in the endoplasmic reticulum,Golgi apparatus and mitochondria,respectively.The ch SNX9 truncated plasmid was also constructed,and the key structural and functional domains interacting with STING were screened by immunoprecipitation technique.In addition,ch STING was overexpressed with ch TBK1 and ch SNX9 in LMH cells,and Co-IP assays were performed to investigate whether ch SNX9 affects the interaction between ch STING and ch TBK1.The results showed that ch SNX9 is localized in mitochondria and Golgi,and the C-terminal functional domain of ch SNX9(270-600aa)can interact with ch STING and act as a regulatory key.Further,ch SNX9 was found to block the binding of STING and TBK1 and thus negatively regulate the c GAS-STING pathway natural immunity.This section further analyzes the molecular mechanism of ch SNX9 to negatively regulate the c GAS-STING pathway during FAd V-4 infection,providing a basis for FAd V-4 prevention and control.In summary,this study identified ch SNX9 as a potential key molecule regulating the c GAS-STING pathway by TMT quantitative proteomics and tandem mass spectrometry,and explored the role of ch SNX9 in the induction of IFN-β production and viral replication by FAd V-4 infection,and clarified that ch SNX9 can block the interaction between STING and TBK1 and thus negatively regulate natural immunity,laying a foundation for further investigation of the immune regulatory mechanism of FAd V-4 infection.