FIG. 5.

Full-length ICP0 stimulates the activity of UbcH5a and UbcH6 in vitro. Wild-type and RING finger mutant full-length ICP0 proteins with N-terminal His-tags were expressed in insect cells using a recombinant baculovirus and purified by nickel affinity chromatography. The left panel shows typical samples of purified His-ICP0 and His-FXE, stained by Coomassie blue. The right panel shows the results of a screen for stimulation of the activity of a panel of E2 ubiquitin conjugation enzymes, probed for high-molecular-weight ubiquitin conjugates using antibody P4D1. Like the GST fusion constructs containing ICP0 RING domains, His-ICP0 stimulates the activity of UbcH5a and UbcH6. The rightmost two tracks show a similar assay using purified RING finger deletion mutant ICP0 (FXE) with UbcH5a and UbcH6.