FIG. 5.

Effect of wt and mutant ICP4 proteins on the recruitment of HA-TFIID complexes to gC promoters. (A) Primary structures of wt and mutant ICP4 proteins. The primary sequence of ICP4 is shown along with a summary of the regions of ICP4 that are similar to those of varicella-zoster virus IE140, with the rectangles indicating similarity (solid rectangles indicate the greatest amount of similarity) (37). Also shown are some of the regions of ICP4 that contribute to various activities, as deduced from genetic and biochemical studies (7, 14, 15, 25, 40, 41, 47, 50, 57). (B) Effect of wt (KOS), n208, and d8-10 ICP4 proteins on HA-TFIID recruitment. HA-TFIID (4 μl) was incubated with 100 fmol of immobilized gC templates in the presence of 75 to 300 fmol (approximately 25 to 75 ng) of purified wt, n208, and d8-10 ICP4 proteins or in the absence of ICP4 in a total volume of 300 μl. As a control for nonspecific binding, HA-TFIID and ICP4 (300 fmol only) were incubated with magnetic resin devoid of immobilized promoter (lanes 5, 9, and 13). After incubation at 30°C, nucleoprotein complexes bound to immobilized promoters were isolated and analyzed as described in Materials and Methods. The presence of ICP4 and HA-TFIID was assessed by Western blot analysis with anti-ICP4 (N15) and anti-HA (anti-HA-TBP) antibodies, respectively. (C) Same as panel B, except that wt and X25 ICP4 proteins were used. The anti-ICP4 antibody used in this experiment (provided by Richard Courtney) allows the detection of the X25 protein.