Fusion to DUb suppresses Gag ubiquitination and ability to release virus. (A) Schematic representation of DUb-Gag fusion proteins. DUb catalytic domain was fused to Gag C-terminal end. (B) DUb fusion to Gag suppresses ubiquitination. 293T cells were transfected with strep-tagged Gag, Gag-DUb or Gag-DUb* fusion proteins (lanes 1, 3, 5, 7, 9, 11; respectively) or in combination with HA-Ub (lanes 2, 4, 6, 8, 10, 12; respectively). Cells and virus pellets were collected 24h post-transfection and incubated with Strep-Tactin beads and captured complexes were probed with the indicated antibodies. (C) Gag-DUb* fusion protein co-assembled with WT Gag and restored the release HIV-1 PTAP-/YP-. 293T cells were transfected with HIV-1 PTAP-/YP- alone (lane 1), or Gag-DUb*Strep (lane 2), Gag-DUbStrep (lane 3), or with HIV-1 PTAP-/YP- and increasing amounts of either Gag-DUbStrep (500 ng, 1 μg or 2 μg) (lane 4, 5, 6) or Gag-DUb*Strep (500 ng, 1 μg or 2 μg) (lane 7, 8, 9). (D) Gag-DUb fusion failed to release virus particles and inhibited HIV-1 release in trans. 293T cells were transfected with HIV-1 alone (1 μg) (lane 1), in combination with either Gag-DUbStrep (1.5 μg) (lane 4), or Gag-DUb*Strep (1.5 μg) (lane 5), or with Gag-DUbStrep or Gag-DUB*Strep alone (1.5 μg) (lanes 2, 3; respectively). Cells and virions were harvested as above and their protein content were analyzed by WB blot using an anti-p24 antibody.