HIV-1-RevNES fails to restore infectivity. (A ) Subcellular localization of GagΔNES and Gag-RevNES in Hela cells analyzed 24h post-transfection with PFV antibodies by confocal microscopy following indirect immunofluorescence in three independent experiments (approximately 200 cells counted each time). (B) Transduction rate of viruses harboring either GagWT, GagG110V, GagΔNES or Gag-RevNES. 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 48h post-transduction. No infectivity was detected in the supernatants of GagG110V, GagΔNES and Gag-RevNES transfected cells in four independent experiments. (C) Western blotting performed on 293T cell extracts and cell-free supernatants shows the absence of viral particles in the supernatants of GagG110V, GagΔNES and Gag-RevNES transfected cells whereas the intracellular Gag mutants are normally produced and matured.