Figure 5

Mapping the functional domains of M2BP.

(A) Schematic representation of M2BP truncation mutants. (B) The HIV-1-producing plasmid indicated was transfected into HEK293T cells with an empty vector (EV), a plasmid expressing M2BP or an M2BP mutant without the signal peptide (M2BPΔSP). In a separate setting, a plasmid expressing M2BP was transiently transfected into HEK293T cells. The M2BP-containing medium was harvested and added to the HIV-1 producing cells (+M2BP), or the M2BP-producing cells were co-cultured with the HIV-1 producing cells (co-culture). The virus containing culture supernatants were collected to infect HeLa-CD4-CCR5 cells. At 48 h postinfection, luciferase activity in the recipient cells was measured (upper panel), and protein levels in the producer cells were analyzed by Western blotting (lower panel). The relative luciferase activity in the absence of M2BP was set as 100. Luciferase activity data presented are means ± SD of three independent experiments, and the Western blotting data are representative of three independent experiments. (C) HIV-1-producing plasmid indicated was transfected into HEK293T cells together with a plasmid expressing the M2BP truncation mutants indicated. At 48 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells and cell lysates were analyzed for expression of the M2BP mutants (lower panel). At 48 h postinfection, luciferase activity was measured in the recipient cells (upper panel). The relative luciferase activity in the absence of M2BP was set as 100. Luciferase activity data presented are means ± SD of three independent experiments, and the Western blotting data are representative of three independent experiments. (D) The HIV-1-producing plasmid indicated was transfected into HEK293T cells with a plasmid expressing the M2BP truncation mutants indicated. A plasmid expressing GFP was included to serve as a control. At 48 h posttransfection, cell lysates and culture supernatants were analyzed by Western blotting. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are means ± SD of three independent experiments. Mock: mock transfected cells; sup: culture supernatant.