Characterization of the GagG110V mutant. (A) Transduction rate of viruses harboring either GagWT or GagG110V. 293T cells were transfected for 48 h with FV vector encoding for GFP together with plasmids expressing Env, Pol and GagWT or GagG110V. Cell free supernatants were used to transduce 293T cells and the viral titer was determined from the number of GFP-positive cells by FACS analysis 48 h post-transduction. No infectivity was detected in the supernatant of GagG110V transfected cells, as observed in five independent experiments. (B) Western blotting performed on 293T cellular extracts and cell free supernatants shows the absence of viral particles in the supernatant of GagG110V transfected cells whereas intracellular GagG110V is normally produced. (C) Electron microscopy revealed, furthermore, the absence of intracellular capsids in 293T cells transfected with GagG110V. Bar: 0.5 μm. (D) Subcellular localization of GagWT and GagG110V in Hela transfected cells with GagWT or GagG110V and analyzed, 24 h post-transfection, by confocal microscopy following indirect immunofluorescence using rabbit polyclonal anti-PFV. GagWT is either nucleocytoplasmic, cytoplasmic or nuclear whereas GagG110V is mainly nuclear, as observed in three independent experiments (approximately 200 cells were counted in each preparation). (E) Western blotting performed on fractionated Hela cell extracts of Gag WT and GagG110V. Detection of the human lactate dehydrogenase (LDH) in cytoplasmic extracts only attests the validity of the fractionation assay (C: Cytoplasm, N: Nucleus).