Fig. 3.

The RING domain of ICP0 is important for its selective activation of AP-1. (a) ICP0 activates AP-1-Luc, but not LEF-Luc or Gal4-Luc reporter genes. ICP0 or control plasmids were co-transfected into 293T cells along with the indicated reporter genes. (ΔN)-β-catenin and Gal4-VP16 are the cognate activators of LEF-Luc and Gal4-Luc, respectively. (b) Schematic diagram of ICP0 is indicated at the top. Positions of the C₃HC₄ Zinc Ring finger domain and the other domains were described. NLS—nuclear localization signal; USP7—sequences required for binding to the ubiquitin-specific protease USP7; ND10—sequences required for efficient localization at ND10 domains. Amino acid sequence of the C₃HC₄ Zinc Ring finger domain (aa 116–156) was shown. The consensus Zinc Ring finger sequence is underlined, and the mutations in this domain are in italics (aa 116 was changed from C to A, aa 136 from H to A and aa 156 from C to A). Diagrams of the ICP0 mutants are shown at the bottom. Black lines indicate the sequences retained in each mutant construct. ICP0 (RM): the Zinc Ring finger domain is site-mutated, ‘X’ indicates the mutations; ICP0 exon1 + 2: N-terminus of ICP0 harboring the RING domain; ICP0 AC and ICP0 MC: C-terminal parts of ICP0 that do not contain the RING domain (see details of these plasmids in Materials and methods). (c) AP-1-Luc activation induced by truncations of ICP0. Flag-tagged-variants of ICP0 were co-transfected into 293T cells along with the AP-1 reporter gene and internal control respectively. 48 h later, cells were collected, and dual-luciferase assay was performed.