Figure 2

The RING finger domain of ICP0 is required for it to negatively regulate IFN-β promoter activity driven by TBK1. (A) A schematic diagram of wildtype ICP0 and its mutant (ICP0 FXE) lacking the RING-finger domain. Schematic created in Paint Shop Pro, version 5.01, https://www.paintshoppro.com. (B) HEK-293T cells were transiently transfected with a reporter construct containing the human IFN-β promoter. Cells were co-transfected with 50 ng of empty vector (EV) control or TBK1 and increasing amounts (10, 50 or 100 ng) of a plasmid encoding the RING finger domain deficient ICP0 (ICP0-FXE). The amount of DNA transfected into the cells was normalised using empty vector (EV). Cells were assayed for reporter gene activity 18 h posttransfection. Results are expressed as the mean ± SD in each case and are representative of three independent experiments expressed as fold stimulation over unstimulated empty vector (EV) control. **p < 0.01 as determined by Student t test. (C) 293T cells were transfected with constructs expressing the empty vector (EV), ICP0, FLAG-tagged TBK1 (lanes 1, 2 and 3) or ICP0 and TBK1 (lanes 4 and 5). Eighteen-hour posttransfection cell extracts were immunoprecipitated with an anti-FLAG antibody. Anti-IgG antibody was used in lane 5 for the control. Immunoprecipitated protein complexes were separated by SDS-PAGE and western blotted using anti-FLAG to detect TBK1 (panel 1) and anti-ICP0 antibody to detect ICP0 (panel 2). Presence of ICP0 was detected in whole cell lysates (WCL) by immunoblotting (panel 3). α-Actinin served as a loading control (panel 4). Results are representative of three independent experiments. The full gel image is included in the supplementary data.