Fig 2: Structure of interferon regulatory factor 9 (IRF9) transcripts. (A) Primate IRF9 transcripts. IRF9 gene structures from human (gene ID: 10379), bonobo (100973587), chimpanzee (744767), baboon (101020807), and Francois' leaf monkey (117069921) are as shown. Light green bars represent exon sequences and dark green bars represent open reading frames (ORFs). The canonical IRF9 ORFs are indicated with green solid squares and primate‐specific IRF9 (PS‐IRF9) ORFs are indicated with solid red square on the right. The accession numbers are listed on the left. The species are listed on the right. The diagrams are directly obtained from the NCBI gene report pages. The scales of the diagrams have been adjusted for comparison purpose among species. (B) Mouse IRF9 transcripts. The mouse (Gene ID:16391) IRF9 gene structure is as shown. The potential exons corresponding to primate IRF9 exons are indicated by dashed blue lines.

Fig 3: ASFV pI215L-mediated degradation of IRF9 is independent of its ubiquitin-conjugating activity. (A) HEK-293T cells cultured in 6-well plates were transfected with empty vector or HA-tagged ASFV wild-type (WT) pI215L or HA-tagged pI215L ubiquitin-conjugating activity defective mutation (C85A, C162A, or C189A). After 24 h, cells were treated with IFN-α (1,000 U/mL) for 8 h. The mRNA levels of ISG15, ISG54, ISG56, and OAS1 were analyzed by qRT-PCR. Data are representative of three independent experiments with n = 3 technical replicates (shown as mean ± SEM). (B) HEK-293T cells in 24-well plates were cotransfected with HA-tagged ASFV pI215L or its mutations along with pISRE-Luc and pRL-TK plasmids. After 24 h, cells were treated with IFN-α (1,000 U/mL) for 12 h, followed by luciferase assays. Data are representative of three independent experiments with n = 3 technical replicates (shown as mean ± SEM). (C) HEK-293T cells were transfected with HA-tagged ASFV I215L or its mutations. After 24 h, cells were treated with IFN-α (1,000 U/mL) for 4 h and collected for immunoblotting analysis. The expression level of the endogenous IRF9 was determined using an anti-IRF9 antibody. ***, P < 0.001.

Fig 4: ISGF3 activity is elevated in BAP1-expressing cells. (A) SDS-solubilized whole cell lysates of UMRC6 cells expressing GFP or a BAP1 construct and control (SCR) or IRF9 shRNAs and blotted with the indicated antibodies (n=3). (B) SDS-solubilized whole cell lysates of UMRC6 cells expressing GFP or a BAP1 construct, treated with the indicated concentrations of ruxolitinib or IFN-⍺2 (2000 IU/ml) for 24 h, and blotted with the indicated antibodies (n=3). For ruxolitinib: +, 0.1 µM; ++, 0.3 µM; +++, 1 µM.

Fig 5: DNA-PK inhibitors hinders M1 virus-induced IFN antiviral response. a Gene ontology analysis of the pathways that are overrepresented among the differentially expressed genes between the M1-treated or vehicle-treated HCT-116 cells. b Cells were treated with vehicle or IFN-β (10 ng/μl) for 16 h. Induction of IRF-9 was analysed by western blot. c–h HCT-116 cells were treated with vehicle or NU7441 (1 μM, NU represent NU7441) and M1 virus (MOI = 1) for 24 h and then processed for the qRT-PCR to measure IFN-stimulated gene transcripts. n = 3. Data represent the mean ± SD. *P < 0.05; **P < 0.01 by one-way ANOVA. i HCT-116 cells were pre-treated with NU7441 (1 μM) for 1 h and then infected with M1-GFP at an MOI of 10 in the presence or absence of recombinant IFNα (1000 units/ml). GFP expression was monitored 48 h after M1 inoculation. Scale bar: 50 μm