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. 2021 Jul 17;297(2):100975. doi: 10.1016/j.jbc.2021.100975

Figure 6.

Figure 6

Impact of unique PPxY1 N- and C-terminal extensions on NEDD4L WW3 binding and HIV budding and infectivity.A, importance of AMOT PPxY1 residues for NEDD4LWW3-dependent release of HIV-1ΔPTAP,ΔYP. Left panels are Western blots showing HEK293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 or indicated mutants (panel 1, anti-AMOT), exogenous FLAG-NEDD4L (panel 2, anti-FLAG) or FLAG- NEDD4LWW3, endogenous GAPDH (panel 3, anti-GAPDH, loading control), and HIV-1 GagΔPTAP,ΔYP and the MA and CA proteolytic processing products (panel 4, anti-MA and anti-CA). Cells were cotransfected with an siRNA-targeting endogenous AMOT and expression vectors for HIV-1ΔPTAP,ΔYP, wt FLAG-NEDD4L (lane 1), FLAG-NEDD4LWW3 (lanes 2–5), and wt or mutant, siRNA-resistant HA-AMOT p130. Right panels show corresponding levels of extracellular, virion-associated CAGag and MAGag proteins (panel 1, anti-MA and anti-CA) and viral titers (panel 2), relative to the value in lane 1, set to 1.0. Numbers within the blots show integrated intensities of the MA band intensities (relative to the value in the control experiment, set to 1.0; average of two independent repeats). Error bars denote the SD from two independent replicates. B, importance of AMOT PPxY1 residues for NEDD4LWW3 binding. Fluorescence polarization binding isotherms and Kd values (μM) for the interaction of WW3 with wt PPxY1 (red), and the E104K/E105K (pink) and A112K (blue) mutants. Error bars denote the SD from three independent replicates. PPxY, Pro-Pro-x (any amino acid)-Tyr.