Figure 5

PRV UL13 inhibits IRF3 binding to IRF3-responsive promoter sequences. A, B PK15 cells were transfected with pcDNA3-Flag (1 μg) or pcDNA3-Flag-UL13 (1 μg) for 24 h and then infected with VSV (1 MOI) (A) or treated with poly(I:C) (10 μg/mL) (B) for 9 h. Whole-cell extracts were subjected to native PAGE. Dimers or monomers of IRF3, UL13, and actin were detected with the respective antibodies. C A549 cells were transfected with pcDNA3-Flag-UL13 or D194A/V195A for 24 h. The transfected cells were treated with poly(I:C) (10 μg/mL) for 9 h. The subcellular location of IRF3 was detected with IRF3 antibody, and UL13 was detected with the Flag antibody. The nuclei were stained with DAPI. D PK15 cells were transfected with pcDNA4-HA-IRF3/5D (2 μg) mixed with pcDNA3-Flag or pcDNA3-Flag-UL13 (1 μg) for 30 h. Whole-cell lysates were immunoprecipitated with the HA antibody or control IgG. The immunocomplexes were then used to detect CBP and IRF3/5D with their respective antibodies. E PK15 cells were transfected with pcDNA3-Flag or pcDNA3-Flag-UL13 (2 μg) for 30 h and then treated with poly(I:C) (10 μg/mL) for 9 h. Whole-cell lysates were immunoprecipitated with the IRF3 antibody or control IgG. The immunocomplexes were then used to detect CBP and IRF3 with their respective antibodies. F Schematic presentation of the IFN-β promoter with the location of IRF3-responsive elements and ChIP primers. G PK15 cells were transfected with pcDNA4-HA-IRF3/5D (5 μg) mixed with pcDNA3-Flag or pcDNA3-Flag-UL13 (2 μg) for 30 h. Chromatin was then immunoprecipitated with the HA antibody or control IgG. The binding of IRF3-responsive elements to the IFN-β promoter was quantified by PCR. H PK15 cells were transfected with pcDNA3-Flag or pcDNA3-Flag-UL13 (4 μg) for 24 h and then treated with poly(I:C) (10 μg/mL) for 9 h. Chromatin was then immunoprecipitated with the IRF3 antibody or control IgG. The binding of IRF3 to IRF3-responsive elements on the IFN-β promoter was quantified by PCR.