Characterization of Phoenix. (A) Immunoblot analysis of the Phoenix products present in the lysates of transiently transfected 293T cells, or in the concentrated supernatant. Cells were transfected with (1) a control plasmid (pCMV-β), (2) an expression vector for Phoenix, or (3) an expression vector for a pro mutant. The membranes were hybridized with an antibody directed against the Gag protein (left) or the Env protein (right). (B) Immunofluorescence study of 293T cells transfected with Phoenix. Gag is marked in red, Env in green, and the nuclei are stained in blue. Note the colocalization of the two proteins, especially at the membrane that separates the two adjacent marked cells. (C) Detection of RT activity in the concentrated supernatant of 293T cells transfected with Phoenix and a series of proviral and control vectors. Concentrated virions were used as a source of RT activity to reverse transcribe synthetic MS2 RNA. Presence of cDNA was revealed via a PCR amplification using appropriate primers. (1) pCMV-β; (2) Phoenix; (3) Phoenix mutated in RT; (4) chimera K109-K115; (5) chimera K109-K113; (6) chimera K109-K108; (7) MLV core proteins).
