Skip to main content

Advertisement

Figure 1 | Retrovirology

Figure 1

From: Ubiquitin conjugation to Gag is essential for ESCRT-mediated HIV-1 budding

Figure 1

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.

Back to article page