DUb-TSG101 interferes with HIV-1 release. (A) Co-expression of DUb-TSG101 inhibits HIV-1 release. 293T cells were transfected with expression plasmids of HIV-1 YP- (lane 1), or co-expressing Flag-TSG101, Flag-DUb-TSG101 or Flag-DUb*-TSG101 (lanes 2, 3, 4, respectively). (B) DUb-TSG101 failed to replace functionally cellular TSG101. 293T cells were transfected twice with RNAi to TSG101 (lanes 2–5) at 36-h intervals. At the second transfection, cells were co-transfected with expression plasmids of HIV-1 YP- alone (lanes 1 and 2) or either Flag-TSG101RR (250 ng) (RNAi Resistant form), Flag-DUB-TSG101 RR or Flag-DUb*-TSG101RR (15 ng) (lanes 3, 4, 5, respectively). Cells and viruses were collected 24 hours post-transfection and their protein content was analyzed by WB using the indicated antibodies. Virus release efficiency was also quantified using HeLa TZM-bl assays from 3 independent experiments and expressed relative to WT HIV (A) or WT TSG101 RR(B). (C) DUb-TSG101 inhibits late steps of HIV-1 budding. Shown are EM images of thin-sectioned 293T cells co-transfected with HIV-1 and DUb-TSG101 (a and b) or with DUb*-TSG101 (c). A high-magnification image of budding virus particles from panel (a) (rectangle) is shown (b) and black arrows indicate particles tethered to the plasma membrane or to each other. Quantification of budding defects was performed and approximately >250 virus particles from 2 independent experiments were examined and categorized as immature budding particles, or mature released particles (±SD).