Mutation of residues located at the tip of Phe105 loop in the Bro1 domain (A) or Alix (B) interfere with their ability to rescue the release of pNL4-3 PTAP-/YP- and pNL4-3 PTAP- mutant viruses, respectively. 293T cells were transfected with pNL4-3 PTAP-/YP- (panel A) or pNL4-3 PTAP- (B) proviral DNA alone (lane 1), with HA-Bro1 domain or HA-Alix (lane 2), with the indicated HA-Bro1 domain (panel A, lanes 3–8) or Alix (B, lanes 3–10) Phe105 loop mutants. Cells and viruses were collected 24h post-transfection and their protein content was analyzed by SDS-PAGE and western blot using an anti-capsid antibody. Expression of Bro1 or Alix and their mutants was detected using an anti-HA antibody. Identical results were obtained in three separate experiments. HIV-1 release ratio (values in percentage) was calculated using the following ratio: release ratio = virus-associated Gag/cell-associated Gag and is shown under panels. C) Release of Alix Phe105 loop mutants was evaluated in 3 independent experiments and expressed relative to the WT Alix (±SD) (n = 3).