Figure 4

UL13 kinase activity is essential for inhibiting IRF3 function. A Schematic presentation of the kinase functional sites of PRV UL13. B The dual-luciferase reporter assay was performed as in Figure 1. The fold activation of luciferase activity is the product of the luciferase activity induced by cGAS–STING with or without PRV UL13 or its kinase-dead mutants, divided by that induced by the empty vector. C PK15 cells were transfected with pcDNA4-HA or pcDNA4-HA-IRF3/5D mixed with pcDNA3-Flag-UL13, or its kinase-dead mutants for 30 h. Whole cells were collected with lysis buffer, and the lysate of the UL13-transfected group was treated with lambda protein phosphatase. HA-tagged IRF3/5D was detected by anti-HA, and Flag-tagged UL13 and its kinase-dead mutants were detected by anti-Flag. D PK15 cells were transfected with pcDNA4-HA or pcDNA4-HA-IRF3/5D mixed with pcDNA3-Flag-UL13, the UL13 kinase-dead mutant or their mixture for 30 h. Whole cells were collected with lysis buffer. HA-tagged IRF3/5D was detected by anti-HA, and Flag-tagged UL13 and its kinase-dead mutants were detected by anti-Flag. E Schematic presentation of the location of gRNAs used to generate UL13 deletion mutants. F PK15 cells infected with 0.1 MOI JSY13 or PRVΔUL13 mutants were harvested at the indicated time points, and virus titre measurements were conducted using the plaque formation assay. G PK15 cells were transfected with or without pcDNA3-Flag-UL13 for 18 h and then infected with 1 MOI of the JSY13 or ΔUL13 mutants for 16 h. Whole cells were collected with lysis buffer. The cell lysates were separated by SDS-PAGE and immunoblotted with an antibody against IRF3, UL42, Flag or actin. H PK15 cells were infected with JSY13 (1 MOI) for 16 h. Whole cells were collected with lysis buffer, and the lysate was treated with lambda protein phosphatase. Endogenous IRF3 was detected with the anti-IRF3 antibody.