Fig 5.

US3 interacts with IRF3. (A) HEK 293T cells were transfected with Flag-tagged US3 plasmid. Twenty-four hours posttransfection, cells were infected with SeV. Flag-tagged US3 was immunoprecipitated (IP) using an anti-FLAG antibody or nonspecific mouse antibody IgG2b (control) and subjected to immunoblot (IB) analysis using an anti-IRF3 antibody to detect endogenous IRF3 interaction. (B) Schematic diagram of the construction of US3-Flag HSV-1. A Flag tag was inserted into the C terminus of US3 in the HSV-1 genome. Confluent HEK 293T cells (C and E) or Vero cells (D) were infected with the indicated viruses at an MOI of 0.1. Growth curves were generated by luciferase activity assays (C and D), and expression of UL42 and UL46 was detected by WB assays (E). (F) HEK 293T cells were infected with WT HSV-1 or US3-Flag HSV-1 for 16 h, and quantitative PCR assays were performed to detect the mRNA levels of IFN-β. Statistical analysis was performed using the Student t test. *, P < 0.05. (G) HEK 293T cells were infected with US3-Flag HSV-1 at an MOI of 5 for 20 h. Co-IP assays were performed to investigate the interaction between endogenous IRF3 and US3. (H) HEK 293T cells were transfected with Flag-tagged US3, US3 K220M, or US3 D305A plasmid. Twenty-four hours posttransfection, co-IP assays were performed to detect the interaction between endogenous IRF3 and US3 K220M or US3 D305A.