FIG 3.

BPLF1 deubiquitinates the Rad6/Rad18 complex. (A) BPLF1 deubiquitinates Rad6/18 in vitro. Ube1 was incubated with Rad6/Rad18 and FLAG-ubiquitin in the presence or absence of BPLF1. Samples were run on a 12% SDS-PAGE gel, immunoblotted, and probed with FLAG, Rad18, and BPLF1 antibody. BPLF1 specifically deubiquitinates Rad6 but not Ube1. Slight deubiquitination of Rad18 was also observed. Input levels are shown at the bottom. (B) Rad18 is likely deubiquitinated by BPLF1 in vivo. 293EBV+ cells were transfected with BPLF1 1-246 and BZLF1 (to induce lytic replication) where indicated. The upper band in the Rad18 panel is consistent with the monoubiquitinated form of Rad18 and appears to be reduced in the presence of BPLF1. BPLF1 and GAPDH loading controls are shown in the middle and bottom panels.