Figure 3

Dominant-negative properties of the GagG110V mutant. (A) Virus titers. Viral particles were produced in the supernatant of 293T cells transfected with the four-plasmid PFV vector system in the presence of increasing amounts of GagHH or GagHHG110V. Target 293T cells were transduced with cell free supernatants and titers were determined by FACS analysis 48 h post-transduction. Viral titers were dramatically reduced following addition of increasing amounts of GagHHG110V. This result is representative of three independent experiments. (B) Western blotting also shows a decrease in the amount of Gag proteins in supernatants whereas they are efficiently produced in 293T cells extracts. Therefore, GagG110V mutant negatively interferes with WT Gag impairing particles production. (C) Co-localization of GagHH and GFP-GagG110V. Hela cells were co-transfected with indicated plasmids and analyzed, 48 h post-transfection, by confocal microscopy following indirect immunofluorescence. GagWT colocalizes with GFP-GagG110V in the nucleus in 80 ± 4% of transfected cells in three independent experiments with approximately 100 cells counted each time. (D) Sequestration of GagWT by GagG110V in the nucleus. Nuclear interaction of GagHH and GFP-GagG110V revealed by co-immunoprecipitation of nuclear extracts of transfected Hela cells, using mouse anti-HA or anti-GFP antibodies followed by western-blotting performed with rabbit polyclonal anti-Gag antibodies (N : nucleus and C : cytoplasm).