Screening Of SAV Proteins Activating F-?B Signal Pathway And Study Of Pathogenic Mechanism

Salmonid Alphavirus infects salmonid fishes and causes pancreatic disease(PD)and sleeping disease(SD).It has spread widely in European farms.The mortality rate is between 60% and 90%,and it has survived.Fish usually grow slowly,causing huge losses to the salmonid fish farming industry.The host infected with the virus first initiates the innate immune response,the pattern recognition receptor(PRR)recognizes the virus,activates the downstream signal transduction pathway,and induces innate immunity by producing inflammatory cytokines,type I interferon(IFN)and other mediators The induction of response,and the NF-?B signaling pathway is one of the mechanisms by which the host exerts an antiviral effect.This study will explore the molecular mechanism of SAV activating the NF-?B signaling pathway,and its key proteins that mediate the inflammatory response,and determine its site of action,and further elucidation of the molecular mechanisms by which SAV proteins interact with host cell proteins to activate the NF-?B signalling pathway to mediate inflammatory responses.Infect CHSE-214 cells with SAV,use the dual luciferase reporter system to verify the ability of SAV to activate NF-?B,and use the dual luciferase reporter system to screen SAV proteins that activate NF-?B and their key functional domains,and upstream pattern recognition Receptor to verify the activation of the NF-?B signaling pathway and the downstream related inflammatory factors produced by the selected protein;analyze the interaction between SAV activator protein and CHSE-214 host cell protein by mass spectrometry,and use CO-IP technology,laser confocal observation,dual luciferase reporter system verify the interaction between virus and host cell,analyze the molecular mechanism of SAV protein activation of NF-?B pathway and its influence on virus replication.The test results are as follows:1.Firstly,the dual luciferase reporter system was used to detect the ability of SAV to activate NF-?B,and it was found that SAV V4640 can significantly activate NF-?B after infection of CHSE-214 cells.With the increase of time and titer of SAV infection of CHSE-214 cells,the dual luciferase activity value was significantly up-regulated,indicating that SAV can activate the NF-?B signaling pathway,and it is time-and dose-dependent with infection time and virus titer.At the same time,we further screened SAV to activate the NF-?B signaling pathway to mediate the key protein of inflammation.It was found that the SAV non-structurally encoded protein Nsp2 has the ability to significantly activate NF-?B,and it is dose-dependent with the concentration of the Nsp2 transfected plasmid,while the activation ability of other proteins is significantly lower than that of Nsp2.2.Using Western Blot analysis,it was found that Nsp2 can cause NF-?B nuclear transfer,p65 phosphorylation,and I?B? degradation at the protein level.Using real-time fluorescent quantitative PCR analysis,it was found that SAV Nsp2 activated the NF-?B signaling pathway and promoted the downstream inflammatory factor IL-6,IFN-1 and Mx1.In order to reversely verify that Nsp2 activates NF-?B to produce downstream inflammatory factors,the use of NF-?B inhibitors during Nsp2 transfection significantly reduces the production of downstream inflammatory factors.The results analyzed the mechanism of non-structural protein Nsp2 causing NF-?B nuclear metastasis and producing downstream inflammatory factors.3.The entire length of Nsp2 was truncated to construct recombinant plasmids pCMV-Nsp2-A,pCMV-Nsp2-B,pCMV-Nsp2-C,pCMV-Nsp2-A1,pCMV-Nsp2-A2,pCMV-Nsp2-B1,pCMV-Nsp2-C1,pCMV-Nsp2-ML,and pCMV-Nsp2-Zf-AD.The NF-?B dual luciferase reporter system was used to accurately locate the key functional domains of NF-?B activated by Nsp2.The results showed that the A2(1-279aa),ML(279-421aa),and Zf(398-495aa)segments of the SAV Nsp2 gene all have functional domains that activate NF-?B.Among them,ML truncated protein is the key functional domain and has the strongest ability to activate NF-?B.4.SAV was infected with CHSE-214 cells,and the Nsp2 recombinant plasmid transfected with CHSE-214 cells was set up as a test group.Real-time fluorescent quantitative PCR was used to detect the TLR signaling pathway in CHSE-214 cells and the expression of key adaptor proteins.The results show that both SAV V4640 and Nsp2 can significantly up-regulate the expression of host cell CHSE-214 pathogen pattern recognition receptors TLR3,TLR7 and TLR8 and adaptor proteins TRAF6 and MAVS.It proves that SAV and Nsp2 can cause an innate immune response in the host and can trigger the body's antiviral defense mechanism.The upstream molecular mechanism of activating host cells is that TLR7 and TLR8 act on TRAF6 protein.RIG-I acts on the MAVS protein to recruit the TRAF6 protein and then acts on the IKK family to activate NF-?B to promote the production of downstream inflammatory cytokines.It shows that SAV V4640 and Nsp2 activate the NF-?B signaling pathway through TLR3,TLR7,TLR8 signaling pathway and RIG-I signaling pathway.5.In order to study whether Nsp2 can affect the replication of SAV,real-time fluorescent quantitative PCR was used to detect the SAV viral load in CHSE-214 cells 24 h,48 h and 72 h after Nsp2 was transfected.The results showed that compared with the control group,the viral load produced by the Nsp2 transfected experimental group at 72 h was significantly suppressed.It is inferred that transfection of Nsp2 to activate the NF-?B signaling pathway produces a large amount of IFN-1 at 72 h,which inhibits the reproduction of SAV.6.Use CO-IP technology to catch the interaction between SAV Nsp2 protein and host cell CHSE-214,and use mass spectrometry to screen out 174 host proteins that interact with Nsp2.Through NCBI blast analysis and annotation,the selected host cell proteins are further used for GO annotation and KEGG metabolic pathway annotation.The results show that according to the GO annotation analysis results,the protein that interacts with Nsp2 among the 174 proteins is involved in animal organ development,biological development,ubiquitin ERAD pathway,ubiquitin mechanism in the endoplasmic reticulum,and stress response to the endoplasmic reticulum,translation,peptide biosynthesis,the production of ribonucleoprotein complexes and other biological processes.The results of KEGG analysis showed that there were 5 proteins related to inflammation.Among them,the Toll-like receptor signaling pathway and the RIG-I signaling pathway related to the NF-?B signaling pathway have enriched protein gene numbers A0A060YTB0(IKK)and A0A2P1K3U9(DDX3),respectively.Furthermore,it is preliminarily determined that the interaction between SAV Nsp2 protein and IKK and DDX3 proteins in the corresponding host cell CHSE-214 can mediate the NF-?B signal transduction pathway.7.Put A0A060YTB0(IKK)and A0A2P1K3U9(DDX3)host proteins into the database for enrichment.According to the analysis of the network results of protein interaction,it can be seen that the A0A2P1K3U9(DDX3)protein in the RIG-? signaling pathway is highly enriched.Further using laser confocal and immunoprecipitation methods to verify the authenticity of the interaction between Nsp2 and DDX3 in CHSE-214 cells.In addition,the test results of the dual luciferase reporter system show that the interaction of DDX3 and Nsp2 will reduce the activity of dual luciferase,and DDX3 will inhibit the activation of NF-?B.It shows that after Nsp2 binds to DDX3,the inhibition of NF-?B by DDX3 is released,thereby activating the signal transduction of NF-?B.In summary,this study confirmed for the first time that SAV can infect host cells and significantly activate the NF-?B signaling pathway,and the non-structural protein Nsp2 protein can activate the NF-?B signaling pathway alone.It was identified that the key domain of non-structural protein Nsp2 to activate NF-?B is located between amino acids 279-421.In addition,it was determined that the signaling pathways mediated by TLR3,TLR7,TLR8,and RIG-? are jointly involved in the activation of NF-?B signaling pathways by Nsp2 and SAV,and Nsp2 can activate NF-?B to produce downstream inflammatory factors IL-6,Mx1,and IFN-1 thereby causing an inflammatory response.Clarified the conduction pathway by which Nsp2 activates the NF-?B signaling pathway.IFN-1 has the ability to inhibit SAV virus replication and reduce the viral load of host cells.Nsp2 activates the NF-?B signaling pathway by interacting with the host DDX3 protein,and participates in the process of viral protein translation and replication.It is speculated that the virus uses the host cell to synthesize its own viral elements,which further indicates that the Nsp2 protein is related to virus replication.Nsp2 plays an important role in SAV replication and pathogenic mechanisms,and provides a theoretical basis for the study of antiviral gene-targeted drugs and disease-resistant breeding.