Structural And Functional Research Of African Swine Fever Virus Protein PA137R

As an acute and fatal infectious disease of swine,African swine fever(ASF)is caused by African swine fever virus(ASFV).Since it was reported in Kenya in 1921,ASFV has caused a devastating effect on the pig industry around the world.In particular,ASFV brought huge losses to the Chinese economy after it was introduced into China in 2018.However,due to the lack of effective commercial vaccines,the prevention and control of ASF has become very difficult and can only be controlled by strict biosafety measures.pA137 R is known to be an important virulence factor encoded by ASFV.Deletion of pA137 R reduced the virulence of ASFV significantly,and induced the production of high levels of antibodies in animals and provide protective immunity for them.Therefore,pA137 R can be a candidate target for vaccine development.However,the research on structure and biological function of pA137 R still exists a large gap.In order to explore the biological function of pA137 R and its role in the course of ASFV infection,the following work was carried out in this study:1.Bioinformatics analysis,expression and purification of pA137 R protein and preparation of pA137 R antibodyThe physicochemical property of pA137 R was analyzed by bioinformatics analysis,it showed that pA137 R is a hydrophilic protein,but there is plenty of disordered loop structure in its protein structure.Hence the stability of pA137 R may be poor.Amino acid sequence alignment among different strains showed that the homology among different strains is high,which is about 90%-100%.Based on this,this research expressed and purified pA137 R protein with prokaryotic expression system and obtained high purity protein.Besides,pA137 R specific antibody with high potency was prepared.2.The subcellular localization of pA137 R and the expression of pA137 R after ASFV infectionThe subcellular localization of pA137 R was observed with laser confocal microscopic,it showed that pA137 R located at endoplasmic reticulum.The expression of pA137 R after infection was analyzed with Western Blot.The results showed that the expression of pA137 R protein could be detected at 8 h after infection and maintained a stable expression level thereafter.Hence the expression of pA137 R may mainly focused on the middle and late stage of viral infection.3.Molecular mechanism of the inhibition of pA137 R on host IFN-Ⅰ responsePrevious studies have shown that the absence of pA137 R can induce protective immunity in the host.Therefore,this study explored the effect of pA137 R as a virulence factor on host innate immunity.Dual-luciferase reporter system and fluorescence quantitative PCR showed that pA137 R inhibited the production of host type Ⅰ IFN(IFN-Ⅰ)significantly.Moreover,it was discovered that pA137 R could interact with an important factor TANK-binding kinase 1(TBK1)in the c GAS-STING pathway through coimmunoprecipitation tandem mass spectrometry.In order to further explore the structural basis and molecular mechanism of pA137 R inhibiting host IFN-Ⅰ response,pA137 R crystals with a resolution of about 15 (?) were obtained through various protein forms such as full-length,truncated and fused with MBP tags and various crystallization methods.But it was not enough to resolve the structure of pA137 R protein.Therefore,the structure of pA137 R was simulated with Alpha Fold 2.The whole structure of pA137 R was similar to a hairpin,including 5 α-helices,4 β-sheets and plenty of disorder loops.The simulated pA137 R structure was used for molecular docking with TBK1,and it was discovered that seven regions of pA137 R,F34-E39,T43-K52,E54-E60,H62-E73,L76-R80,L89-T93 and A115-K131 participated in the interaction between pA137 R and TBK1.Further investigation through dual-luciferase reporter system and structural analysis revealed that pA137 R may inhibit the kinase activity of TBK1 through three key regions of pA137 R,H62-E73,L76-R80 and L89-T93,thus inhibiting the host IFN-Ⅰ response.In conclusion,this study explored the structure and biological functions of pA137 R.It was discovered that pA137 R could inhibit the production of host IFN-Ⅰ,and the molecular mechanism of this inhibition was explored based on the structure analysis,which provided a theoretical basis for the precise design of ASFV vaccine.