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. 2019 Feb 5;93(4):e01934-18. doi: 10.1128/JVI.01934-18

FIG 6.

FIG 6

VP23 disrupts the association between TBK1 and IRF7 by interacting with IRF7. (A and B) HEK293T cells were cotransfected with the VP23-Flag and IRF7-HA expression plasmids for 36 h, followed by a coimmunoprecipitation (co-IP) assay for VP23-Flag and IRF7-HA using anti-HA (IP: HA) (A) or anti-Flag (IP: Flag) (B) antibody. (C) DF-1 cells were transfected with the VP23-Flag expression plasmid or an empty vector, and at 36 h posttransfection, a coimmunoprecipitation assay was performed with anti-Flag antibody. (D) CEFs were left uninfected or infected with MDV at an MOI of 0.1, and at 48 h postinfection, coimmunoprecipitation was performed with the indicated antibodies. (E) DF-1 cells were transfected with VP23-Flag and/or IRF7-HA expression plasmids for 24 h and then fixed and processed for dual labeling. IRF7 (green) and VP23 (red) proteins were visualized by immunostaining with rabbit anti-HA and mouse anti-Flag antibodies. Cell nuclei were counterstained with DAPI (blue). The areas of colocalization in merged images are shown in yellow. (F and G) DF-1 cells were cotransfected with TBK1-HA and IRF7-Flag expression plasmids with or without different amounts of VP23-Myc. After 36 h of transfection, cell extracts were analyzed by immunoprecipitation (IP) using anti-HA antibody (IP: HA) (F) or anti-Flag antibody (IP: Flag) (G). (H) Schematic representation of the full-length IRF7 (aa 1 to 492) and different truncated IRF7 proteins, including IRF7DBD (aa 1 to 143), IRF7△DBD (aa 143 to 492), IRF7AD (aa 143 to 303), and IRF7IRD (aa 303 to 492). The various domains of IRF7 include the DNA-binding domain (DBD), constitutive activation domain (CAD), virus-activated domain (VAD), inhibitory domain (ID), and signal response domain (SRD). (I) Full-length IRF7-Myc or the truncated IRF7-Myc was transfected with VP23-Flag into DF-1 cells. After 36 h, the cells lysates were immunoprecipitated with anti-Flag and analyzed by Western blotting. (J) Full-length IRF7-Myc or the truncated IRF7-Myc was transfected with TBK1-HA into DF-1 cells. After 36 h, the cell lysates were immunoprecipitated with anti-HA and analyzed by Western blotting. The data represent the results from one of the triplicate experiments. Statistical analysis was performed using Student's t test (**, P < 0.01).