Molecular Mechanism Of Duck Tembusu Virus Protein Regulating STING,MDA5,TLR4-mediated Immune Response

Innate immunity is the host’s first line of defense against viral infection.Type I interferon(Interferon,IFN)plays an important role in innate immunity.Flavivirus RNA can be recognized by Toll-like receptors(Toll-like receptors,TLRs)or RIG-like receptors(Retinoic acid-inducible gene I-like Receptors,RIRs)in the cytoplasm,and further activate related signaling pathways,induce downstream anti-viral molecules production.There are three key adapter molecules downstream of the pattern recognition receptors,namely MAVS(Mitochondrial antiviral signaling),STING(Stimulator of interferon gene),and TRIF(TIR domain-containing adapter molecule 1).Most flaviviruses can target one or more adapter molecules to suppress host innate immunity.There is a game between the virus and the host.On the one hand,the host’s innate immune system can recognize and respond to viral infection.On the other hand,the virus has evolved various ways to inhibit the activation of the host’s innate immune system to establish infection.Tembusu virus(TMUV)belongs to the family of Flaviviridae and the genus Flavivirus.Virus particles are mainly composed of envelope,nucleocapsid,and single-stranded positive-strand RNA.At present,TMUV antagonizes the activation of duck innate immunity and its internal mechanism is still unclear.In order to elucidate the regulation mechanism of the virus on the innate immunity of ducks during TMUV infection,the following works were carried out:(1)TMUV NS2B3 cleaves STING to antagonize the activation of innate immune pathways.Duck embryo fibroblasts(DEFs)were infected with TMUV.The results showed that the expression of IFNβ could not be significantly up-regulated when TMUV replicated in large numbers(P > 0.05).Further research found that overexpression of NS2B3 protein can inhibit the production of IFNβ mediated by RIG-I,MDA5(Melanoma differentiation-associated protein 5),MAVS,and STING,but can’t inhibit the downstream TBK1(TANK-binding kinase 1)and IRF7(Interferon regulatory factor 7)mediate production of IFNβ,indicating that NS2B3 may target STING or its upstream protein,thereby antagonizing the host IFN signaling pathway.In order to identify the interaction mode between NS2B3 protein and STING protein,BHK-21 cells were co-transfected with STING and NS2B3 eukaryotic expression plasmids,and the results of Western blot showed that an extra band appeared at about 10 k Da below the STING protein band,but after mutating the NS2B3 protease active site,the STING protein was not cleaved.The above results indicated that NS2B3 protein can make use of its protease activity to cleave STING protein.Further,in order to determine how the STING protein affects NS2B3 cleavage,alanine scanning mutations were performed on the STING protein.The results of Western blot showed that the mutations at STING R84 and G85 not only seriously affected NS2B3 cleavage,but also the ability of NS2B3 antagonized STING-mediated IFNβ and downstream interferon stimulation genes(Interferon stimulation genes,ISGs)was significantly weakened(P < 0.01).Particularly important,co-immunoprecipitation results showed that if NS2B3 can’t bind to the truncated STING protein(retaining the cleavage site),then NS2B3 no longer cleaves STING.In summary,it can be concluded that the interaction between NS2B3 and STING proteins is a prerequisite for NS2B3 to cut STING.NS2B3 and STING were truncated and expressed,and the binding regions of NS2B3 and STING were found to be located in the amino acid 221-225 region of STING and the NS2 B region of NS2B3 protein,respectively.Therefore,this study identified the mechanism by which TMUV NS2B3 targets duck STING protein to escape the innate immune system,providing a new model for studying the interaction between flavivirus NS2B3 and STING protein.(2)TMUV NS2 B antagonizes the activation of innate immunity by inducing MDA5 protein degradation.Since TMUV protein may inhibit IFNβ production by targeting multiple host proteins,this paper explored the interaction between other TMUV proteins and IFN signaling pathway in parallel.First,TMUV-infected DEFs were transfected with poly(I:C).Quantitative PCR results showed that TMUVinfected DEFs could inhibit poly(I:C)-induced IFNβ production.Next,in order to identify poly(I:C)-specific activation of signaling pathways in DEFs,DEFs knock-down RIG-I or MDA5 were transfected with poly(I:C).Quantitative PCR results showed that poly(I:C)-induced IFNβ expression in MDA5-knockdown DEFs could not be significantly induced(P < 0.01).The above indicated that TMUV may antagonize host innate immunity by targeting MDA5 or its mediated pathway.Further studies found that TMUV infection of DEFs could specifically target endogenous MDA5 and lead to its degradation.Different types of inhibitors(protease inhibitors,autophagy inhibitors,apoptosis inhibitors)were used to screen the pathway of TMUV-induced MDA5 degradation,and the results showed that the autophagy inhibitor 3-MA could partially restore the expression of MDA5.Pretreatment of 3-MA before infection with TMUV can increase the expression of MDA5,and the increased expression of MDA5 can promote the activation of type I IFN pathway,which in turn inhibits TMUV replication.Next,we studied the molecular mechanism of TMUV promoting the degradation of MDA5 through autophagy.Western blot results showed that NS2 B can degrade MDA5 in a dose-dependent manner.The results of screening the region of NS2 B responsible for inducing MDA5 degradation showed that: NS2 B lacking the hydrophilic region(deletion of amino acids 51-92)no longer induced MDA5 degradation,and the ability of NS2 B to inhibit the production of IFNβ was significantly weakened(P < 0.01).In summary,this chapter found that TMUV infection can induce autophagy in cells,NS2 B can bind MDA5 to promote its degradation by autophagy,and finally lead to the inhibition of the activation of innate immunity.(3)Escherichia coli LPS(Lipopolysaccharide)and its receptor TLR4(Toll-like receptor 4)are involved in TMUV adsorption.In order to explore the possibility of TMUV gastrointestinal infection,TMUV was used to infect 2-day-old ducklings by gavage,and the infectious virus could be detected in the duck brain and kidney 24 hours later,indicating that TMUV can infect through the digestive tract.In order to investigate the effect of gastrointestinal microorganisms on TMUV oral infection,we pretreated ducklings with antibiotics,and then TMUV was used to infect ducks by gavage.The results showed that antibiotic pretreatment would significantly reduce the incidence of TMUV in ducklings intestinal proliferation(P < 0.01).This study continued to use virus adsorption experiments to further explore the interaction between Gram-negative bacterial outer membrane LPS and TMUV,and the results showed that LPS can promote the adsorption of TMUV on DEF.As the most important receptor on the surface of LPS cells,TLR4 can transmit the cell signal activated by LPS.We found that TMUV E protein has the same cellular localization as TLR4,and the two can specifically bind.DEFs were pretreated in three different ways: TLR4 polyclonal antibody,purified TLR4 protein,and TLR4 endogenous expression knockdown.Adsorption tests showed that these three ways could inhibit TMUV invasion.Heterologous expression of duck TLR4 on non-duck-derived cells(BHK-21 cell line)promoted TMUV infection,and in DEFs,a TLR4 inhibitor(TLR4-RS)could significantly inhibit LPS-promoted TMUV proliferation(P < 0.01).In order to further analyze the regulation of TLR4-mediated cell signaling pathways by TMUV E,in this study,LPS was used to stimulate TMUV-infected DEFs,and it was found that TMUV could compete with LPS for binding to TLR4,thereby down-regulating the activation of inflammatory signaling pathways induced by LPS(P < 0.0001).In summary,this study first established the positive role of the innate immune PRR TLR4 in the proliferation of flaviviruses and analyzed its molecular mechanism,which provided important experimental data for the theory of non-mosquito vector transmission of flaviviruses.In summary,this dissertation systematically elucidates the molecular mechanism of TMUV escaping duck innate immunity through the study of the interaction between TMUV protein and host innate immune molecules.