African Swine Fever Virus Non-structural Protein A238L Activates The TBK1-IRF3 Signaling Pathway Of Infection Immunity

African swine fever virus(ASFV)is a double-stranded DNA virus with an envelope.The typical symptoms of African swine fever(ASF)include high fever,bleeding,anorexia,diarrhea,constipation,and cyanosis.The latest reports indicate that ASFV variants have diverse clinical symptoms such as a high lethality and fast epidemic rate and are difficulty to control.ASFV has almost the largest genome among all DNA viruses,the mechanisms of immune evasion are complex.Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design.The innate immune system plays an important role in defensing against invading microbial pathogens.cGAS recognizes cytoplasmic DNA,activated cGAS binds to STING on the endoplasmic reticulum.RIG-I recognizes cytoplasmic RNA,leading to RIG-I binding to MAVS on the mitochondria.The cGAS-STING and RIG-I-MAVS pathways both induce TBK1 and IRF3 phosphorylation.Activated IRF3 translocates to the nucleus and induces the transcription of type I interferon(Interferon,IFN).Poly(dA:dT)as DNA virus mimic and poly(I:C)as RNA virus mimic activate IRF3 and induce IFN expression.As the largest DNA virus,ASFV encodes numerous non-structural genes.Non-structural proteins such as DP148R,DP96R and K205R are involved in regulating the antiviral immune response of host cells.A238L,a non-structural protein of ASFV,binds to the amino terminal region of acetyltransferase p300/CBP and inhibits the innate immune response mediated by NF-κB.However,whether A238L regulates IRF3-mediated infection immunity has not been reported.We retrieved the ASFV full genome sequence from the GenBank database,designed A238L primers,and used DNA extracted from the spleen tissue of ASFV-infected pigs as a template to amplify the A238L full gene fragment by PCR.The PCR product was cloned into pcDNA3.1-3×Flag.Plasmid DNA was transfected into three different cell lines(L929,mouse fibroblasts;IPEC-DQ,porcine intestinal epithelial cells;3D4/21,porcine macrophages).A238L was expressed in all three cell lines with the anticipated molecular weight,indicating that the A238L vector was successfully constructed.We first investigated whether A238L affects the activation of IRF3.We employed the above three cell lines to transfect the A238L expression vector,and analyzed the phosphorylation of TBK1 and IRF3 by Western blot(WB).We found that the phosphorylation levels of TBK1 and IRF3 in the cells transfected with the A238L gene were significantly higher than those in the control cells transfected with the empty vector.In cells transfected with the A238L gene,poly(dA:dT),poly(I:C),human simplex virus 1(HSV-1)and Sendai virus(SeV)stimulation further increased the phosphorylation levels of TBK1 and IRF3.Consistent with previous reports,A238L inhibits the phosphorylation levels of IKK and p65 subunit in the NF-κB signaling pathway.Luciferase reporter assay showed that A238L inhibited the expression of luciferase reporter gene driven by NF-κB.Indirect immunofluorescence staining revealed that the number of IRF3-positive cells in the nucleus was increased significantly in A238L-transfected L929 cells.WB revealed that the level of pIRF3 in the nuclear component of the cell lysate transfected with A238L was also significantly higher than the level of pIRF3 in the control cells transfected with the empty vector.Luciferase reporter assay revealed that A238L can activate IRF3 promoter activity in the presence of poly(dA:dT),poly(I:C),HSV-1 or SeV.Similar observations were made with the IFN-β promoter-drive luciferase reporter gene.Fluorescence quantitative RT-PCR revealed that the expression levels of IFN-β and interferon stimulating gene(ISG)were significantly higher L929 cells transfected with A238L plasmid than those transfected with empty vectors.A238L also enhanced poly(dA:dT)or poly(I:C)-induced IFN-β and ISG expression.We further studied the antiviral activity in the conditioned media of A238L-transfected L929 cells.The conditioned media from L929 cells transfected with A238L and/or poly(dA:dT),was added it to cells infected with Sendai virus(carrying green fluorescent protein,GFP),virus replication was analyzed by flow cytometry.The conditioned media of A238L-transfected cells inhibited Sendai virus replication more potently than the control;poly(dA:dT)plus A238L inhibited Sendai virus replication more profoundly.Finally,we tested the effect of A238L on the expression of TBK1 acetylated protein.The results show that A238L enhances the activation of the TBK1-IRF3 pathway and promotes the antiviral immune response by down-regulating TBK1 acetylation.In summary,our study shows that A238L induces IFN-β and ISG expression and inhibits virus replication by promoting the phosphorylation of TBK1 and IRF3.Contrary to the function of most non-structural genes of viruses,although A238L can inhibit the activity of NF-κB,it promotes antiviral innate immunity.