Fusion of the UL36 catalytic domain DUb to TSG101 inhibits ESCRT-I ubiquitination. (A) Schematic representation of the DUb-TSG101 fusion proteins. DUb and DUb* domains were fused to TSG101 as depicted. (B) Effect of DUb fusion to TSG101 on ESCRT-I ubiquitination. 293T cells were transfected with ESCRT-I members [Flag-tagged VPS28 (800 ng), VPS37 (1.7 μg) and MVB12 (800 ng)] and either strep-TSG101 (2.5 μg) (lanes 1 and 7), DUb-TSG101 (lanes 3 and 9) or DUb*-TSG101 alone (600 ng) (lanes 5 and 11) or with HA-Ub (1.5 μg throughout the study unless otherwise specified) (lanes 2, 4, 6, 8, 10, 12). Immunocomplexes were analyzed by western (WB) blotting (WB) using the indicated antibodies. (C) DUb-TSG101 fusion proteins bind HIV-1 NCp6 region. GST (lanes 1, 3 and 5) and GST-NCp6 (lanes 2, 4 and 6) were captured on beads and then incubated with lysates from 293T cells expressing Flag-ESCRT-I/Tsg101 (lane 2), DUb-TSG101 (lane 4) or DUb*-TSG101 (lane 6). Captured proteins and cell lysates were analyzed by WB using the anti-Flag antibody. GST fusion proteins were visualized by Coomassie blue staining. (D) DUb-TSG101 deubiquitinated Gag assembly complexes. 293T cells were co-transfected with HIV-1 YP- mutant (1 μg throughout the study unless otherwise specified) and HA-Ub alone (lanes 1, 4 and 7), with strep-DUb-TSG-101 (lanes 2, 4 and 6) or strep- DUb*-TSG-101 (lanes 3, 6 and 9). Insoluble Gag-enriched fractions were isolated and solubilized to Strep-Tactin-capture DUb-TSG101 or DUb*-TSG101 containing complexes. Gag proteins associated with these complexes and their ubiquitination status assessed by antibodies to p24 and HA, respectively, and input fractions were probed with the indicated antibodies.