Figure 3. AMOT p130 stimulates NEDD4L-dependent release of HIV-1_{ΔPTAP, ΔYP}.

Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins (panel 1, anti-AMOT), exogenous FLAG-NEDD4L (panel 2, anti-FLAG), endogenous GAPDH (panel 3, anti-GAPDH, loading control) and HIV-1 Gag_{ΔPTAP, ΔYP} proteins (panel 4, anti-MA and anti-CA). Cells were co-transfected with expression vectors for HIV-1_{ΔPTAP, ΔYP} (lanes 1–6), FLAG-NEDD4L proteins (lanes 3–6, with wild type (WT) FLAG-NEDD4L in lanes 3 and 4 and FLAG-NEDD4L_ΔWW, in lanes 5 and 6), and wild type HA-AMOT p130 (lanes 2, 4 and 6) or an empty vector control (lanes 1, 3, and 5). Right panels show corresponding levels of extracellular, virion-associated CA^Gag and MA^Gag proteins (panel 1, anti-MA and anti-CA) and viral titers (panel 2, IU denotes ‘infectious units’). Here and in subsequent figures: (1) error bars denote standard deviations between independent repetitions of the experiment, n = 5 in this case, and (2) numbers within the blots show integrated intensities of the CA band intensities (relative to the value in the control experiment, set to 1.0). Here and throughout significance is denoted by: NS, not significant (p > 0.05); *0.05 > p > 0.01; **p < 0.01.

DOI: http://dx.doi.org/10.7554/eLife.03778.008

Figure 3—figure supplement 1. Dose-dependent AMOT p130 stimulation of NEDD4L-dependent release of HIV-1_{ΔPTAP, ΔYP}.

Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins (panel 1, anti-AMOT), exogenous FLAG-NEDD4L (panel 2, anti-FLAG), GAPDH (panel 3, anti-GAPDH, loading control) and HIV-1 Gag_{ΔPTAP, ΔYP} proteins (panel 4, anti-MA and anti-CA). Cells were co-transfected with expression vectors for HIV-1_{ΔPTAP, ΔYP} (lanes 1–8), FLAG-NEDD4L (lanes 2–8), and increasing quantities of an AMOT p130 expression construct (lanes 3–8, 0.25–1.5 μg DNA). Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant (3 μg DNA total). Right panels show corresponding levels of extracellular virion-associated Gag proteins (upper panel, anti-MA and anti-CA) and viral titers (lower panel), n = 3.

DOI: http://dx.doi.org/10.7554/eLife.03778.009

Figure 3—figure supplement 2. The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1_{ΔPTAP, ΔYP}.

Left panels are western blots showing 293T cellular levels of endogenous AMOT, exogenous p80 and exogenous HA-AMOT p130 proteins (panel 1, anti-AMOT), exogenous FLAG-NEDD4L (panel 2, anti-FLAG), GAPDH (panel 3, anti-GAPDH, loading control) and HIV-1 Gag_{ΔPTAP, ΔYP} proteins (panel 4, anti-MA and anti-CA). Cells were co-transfected with expression vectors for HIV-1_{ΔPTAP, ΔYP} (lanes 1–6), AMOT p80 (lanes 2 and 5), AMOT p130 (lanes 3 and 6) and FLAG-NEDD4L (lanes 4–6). Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant (2.5 μg DNA total). Right panels show corresponding levels of extracellular virion-associated Gag proteins (upper panel, anti-MA and anti-CA) and viral titers (lower panel), n = 4.

DOI: http://dx.doi.org/10.7554/eLife.03778.010