Fig 2: The A137R protein (pA137R) promotes the autophagy-mediated lysosomal degradation of TBK1. (A and B) pA137R decreases TBK1 expression. (A) HEK293T cells were transfected with pMyc-A137R (0, 0.25, 0.5, or 1 µg), and subjected to isolation of cellular total mRNAs followed by quantification of TBK1 transcription level by reverse transcription-quantitative PCR. (B) HEK293T cells were cotransfected with p3’Flag-TBK1 (1 µg) together with pMyc-A137R (0, 0.25, 0.5, or 1 µg), and subjected to isolation of total cellular proteins followed by analysis by Western blotting. (C) The A137R gene-deleted ASFV mutant (ASFV-?A137R) resulted in higher TBK1 expression in primary porcine alveolar macrophages (PAMs) than did the wild-type ASFV HLJ/18 strain (ASFV-WT). PAMs were infected with ASFV-?A137R or ASFV-WT for 24 hours post-infection (hpi) at a multiplicity of infection (MOI) of 1, and the expression of TBK1 or pA137R was analyzed by Western blotting using the indicated antibodies. (D) Viability of HEK293T cells treated with the indicated inhibitors. HEK293T cells were grown for 24 h and then treated with dimethyl sulfoxide (DMSO [negative control]) (0, 10, 100, or 200 µM), MG132 (0, 5, 10, or 20 µM), bafilomycin A1 (BafA1) (0, 50, 100, or 200 nM), or 3-methyladenine (3-MA) (0, 5, 10, or 20 mM) for 8 h. The cell viability was determined as described above. (E) HEK293T cells were cotransfected with p3×Flag-TBK1 and pMyc-A137R or pCMV-Myc for 24 h and then treated for 8 h with DMSO (negative control), MG132 (20 µM), BafA1 (100 nM), or 3-MA (10 mM), respectively. The expression of TBK1 or pA137R was analyzed as described above. GAPDH was used as a loading control. (F) pMyc-A137R and p3×Flag-TBK1 were cotransfected into autophagy-related protein 5 (ATG5)-knockout or wild-type (WT) HeLa cells for 36 h, and the expression of TBK1 or pA137R was examined with the indicated antibodies as described above. GAPDH was used as a loading control. Error bars denote standard errors of the means. All the data were analyzed using Student's t test. ns, not significant.

Fig 3: The A137R protein (pA137R) inhibits the beta interferon (IFN-β) signaling pathway through targeting TBK1. (A to D) HEK293T cells were cotransfected with pIFN-β-Fluc expressing firefly luciferase (0.05 μg), pTK-Rluc expressing Renilla luciferase (0.01 μg), and p3×Flag-cGAS (A), -STING (B), -TBK1 (C), or -IRF3-5D (the activated form of IRF3) (D), along with pMyc-A137R or pCMV-Myc, and the luciferase activities were measured at 24 hours post-infection. The expression of cGAS, STING, TBK1, IRF3-5D, or pA137R was analyzed by Western blotting using mouse anti-Flag or -Myc monoclonal antibody. GAPDH was used as a loading control. Error bars denote standard errors of the means. All the data were analyzed using Student's t test: ***, P < 0.001; ns, not significant.

Fig 4: The A137R protein (pA137R) interacts with TBK1. (A) HEK293T cells were transfected with p3×Flag-cGAS, -STING, -TBK1, or -IRF3 for 48 h and lysed with NP-40 buffer. The purified GST or GST-pA137R protein was used to pull down the key adaptors of the cGAS-STING pathway in the lysates and analyzed by Western blotting using mouse anti-GST or -Flag monoclonal antibody (MAb). (B) HEK293T cells were cotransfected with p3×Flag-TBK1 and pMyc-A137R for 24 h and then incubated with rabbit anti-Flag or mouse anti-Myc MAb and Alexa Fluor 488 (green)- or 633 (red)-conjugated secondary antibody, respectively. Cell nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI). Bars, 10 µm. (C) Primary porcine alveolar macrophages (PAMs) were infected with the wild-type ASFV HLJ/2018 strain (ASFV-WT) at a multiplicity of infection (MOI) of 1 for a coimmunoprecipitation assay. The lysates were collected at 48 hours post-infection and incubated with protein G agarose, along with rabbit anti-pA137R polyclonal antibodies (PAbs) or irrelevant rabbit immunoglobulin G (IgG), and then the bound proteins were analyzed by Western blotting using in-house mouse anti-pA137R or rabbit anti-TBK1 PAbs. (D) PAMs were uninfected or infected with ASFV-WT for 24 h at an MOI of 1, and the localization of pA137R and TBK1 was visualized by laser confocal microscopy using in-house mouse anti-pA137R or rabbit anti-TBK1 PAbs and the indicated secondary antibody, respectively. Cell nuclei (blue) were stained with DAPI as described above. Bars, 10 µm.

Fig 5: Serine/threonine-protein kinase TBK1 antibody screening by immunoblot.Lysates of U2OS (WT and TBK1 KO) were prepared and 50 µg of protein were processed for immunoblot with the indicated TBK1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier, except for antibodies 28397-1-AP, PA5-17478, 703154, ab40676, and ab109735, which were titrated at 1/10000 because the signal was too strong following the supplier’s recommendations. Antibody dilution used: NB100-56705 at 1/500; 28397-1-AP at 1/10000; 67211-1-Ig at 1/1000; PA5-17478 at 1/10000; 703154 at 1/10000; ab12116 at 1/600; ab40676 at 1/10000; ab109735 at 1/10000; GTX113057 at 1/2000, 38066 at 1/1000, 3504 at 1/1000. Predicted band size: ~83 kDa.