Nedd4-2s mediated Gag ubiquitination correlates with virus release. (A) Gag fusion with DUb inhibits virus budding. EM images of thin-sectioned cells co-transfected with HIV-1 and Gag-DUb (a) or Gag-DUb* (b). Insets show high-magnification images of a budding or released virus particle and black arrows indicate arrested assembly sites (a) or virions (b). Quantification of budding defects was performed as described in Figure 2. (B) Nedd4-2s-mediated Gag-ubiquitination relieves DUb-TSG101 inhibitory effect on HIV-1 release. Cells were transfected with HA-Ub (lane 1) or HIV-1 YP- alone (lane 2), or with both alone (lane 3), in combination with Strep-DUb-TSG101 (200 ng) (lanes 4) or with the 3 precedent plasmids and Nedd4-2s (150 ng) (lane 5). Cells and virus were harvested after 24 h; cell lysates were incubated with Strep-Tactin beads and the complexes (upper panel), virus pellets (middle panel) and input fractions (lower panels) were probed with the indicated antibodies. (C) Nedd4-2s ubiquitinates Gag at the membrane. Cells expressing HIV YP- were co-transfected with either HA-Ub alone (lane 1), with DUb-TSG101 alone (lane 2) or in combination with Nedd4-2s (lane 3). The P100 fractions of each sample were either analyzed by WB using anti-p24CA antibody (second panel from top) or incubated with anti-HA antibody coated beads. Eluates were analyzed by WB with anti-p24CA antibody to detect ubiquitinated Gag molecules. (D) Nedd4-2s failed to relieve the inhibitory effect of DUb-Gag. 293T cells were transfected with HIV-1 (1 μg) (lane 1), Gag-DUb*Strep (1.5 μg) (lane 2) or with both (lane 3), with Gag-DUbStrep alone (1.5 μg) (lane 4) or with increasing amounts of Nedd4.2s (100 and 200 ng) (lanes 5 and 6). Cells were also transfected with Gag-DUbStrep in combination with HIV-1 (lane 7) or with increasing amount of Nedd4-2s (lanes 8 and 9). Triangles indicate lanes where increasing amounts of Nedd4-2s are expressed.