The Nucleocapsid Region of HIV-1 Gag Cooperates with the PTAP and LYPXnL Late Domains to Recruit the Cellular Machinery Necessary for Viral Budding
Figure 7
Over-expression of Bro1-V inhibits HIV-1 budding in a CHMP4–dependent manner.
(A) The inhibitory effect of Bro1-V is dependent on its CHMP4 binding site. 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated mutants of Bro1-V. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Bro1-V but not Bro1-VI212D co-immunoprecipitates with HIV-1 GagΔp6. 293T cells were co-transfected with GagΔp6-Pol along with expression vectors for HA-Alix, HA-Bro1-V, HA-Bro1-VI212D and HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (C–D) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Bro1-V. (C) The black boxes indicate two regions of interest shown in (a) and (b) at a greater magnification. (D) HIV-1 arrested budding structures. Arrows indicate the electron-dense “ring-like” structure visible at the budding neck of arrested particles.