Fig 2: Downregulation of endogenous M2BP enhances HIV-1 virion production.A control shRNA (Ctrli) or an shRNA targeting M2BP (M2BPi) was stably expressed in HEK293T cells. (A) M2BP protein levels were measured by Western blotting. (B–E) HIV-1-producing plasmids indicated were transiently transfected into cells with (rescue) or without (EV) a rescue M2BP-expressing plasmid. A plasmid expressing GFP was included to serve as a control for transfection efficiency and sample handling. (B,C) At 48 h posttransfection, equal volumes of culture supernatants were used to infect HeLa-CD4-CCR5 cells. At 48 h postinfection, luciferase activity was measured. The relative luciferase activity in the cells infected with the virus produced in control cells was set as 100. Data presented are means ± SD of three independent experiments. (D,E) The producer cells and culture supernatants were harvested for Western blotting analyses. The intensities of the p24CA bands in culture supernatants and the p55Gag bands in cell lysates were quantified with the Image J software. The p24CA release level was calculated as the p24CA level in the culture supernatant divided by the p55Gag level in the cell lysate. The relative p24CA release level in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. **p < 0.01

Fig 3: M2BP inhibits both HIV-1 Env processing and virion production.(A–D) An empty vector (-) or increasing amounts of a plasmid expressing M2BP-myc was transfected into HEK293T cells together with the HIV-1-producing plasmids indicated. A plasmid expressing GFP was included to serve as a control for transfection efficiency and sample handling. (A,B) At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes (middle panel) or equal amounts of CA-containing culture supernatants (lower panel) were used to infect HeLa-CD4-CCR5 cells. At 48 h postinfection, luciferase activity was measured in the recipient cells. Relative specific infectivity was defined as the luciferase activity in the recipient cells divided by the amount of the CA-containing virus used to infect the cells. The relative CA protein level, luciferase activity and specific infectivity in the absence of M2BP were set as 100. Data presented are means ± SD of three independent experiments. (C,D) The virions in culture supernatants were pelleted by ultracentrifugation. Protein expressions in the producer cells, culture supernatants and virion pellets were analyzed by Western blotting. Mock: mock transfected cells. (E) The plasmids indicated were transfected into HEK293T cells with (+) or without (-) a plasmid expressing M2BP. A plasmid expressing GFP was included to serve as a control. At 48 h posttransfection, cells and culture supernatants were harvested for Western blotting analyses.

Fig 4: 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.

Fig 5: M2BP mediates the interaction between Gag and VIM.(A–C) HEK293T cells were transfected with plasmids expressing the proteins indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with antibodies indicated, followed by Western blotting analyses. (D) Hela cells were transfected with plasmids indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting analyses. (E) HeLa cells were transfected with pVIM-TagBFP, together with an empty vector or a plasmid expressing M2BP-mKO. At 36 h posttransfection, cells were transfected again with pHIV-1-iGFPΔEnv for 36 h. Cells were fixed in paraformaldehyde. Fluorescence images were obtained by confocal fluorescence microscopy.