Mutation of specific serine residues in the Vpu cytoplasmic tail cripples its ability to deplete CD4 and CD317 and to promote HIV-1 release. 293TCD4 cells were transfected to express HIV-1Δnef Δvpu GFP and HA-CD317 together with either Vpu wt ("Vpu") or Vpu mutants, in which S52 or/and S56 were replaced by alanine. (A) Two days post-transfection, the yield of infectious HIV-1 in the supernatant and cell-associated levels of Vpu, HA-CD317, p24CA, and MAPK were analyzed. Western blots shown represent samples run on the identical gel with gaps indicating areas where non-informative lanes were omitted. The HIV-1 yields are plotted relative to the condition in the absence of HA-CD317 (Control), which was set to 100%. Shown are arithmetic means + SD (n = 6) from one of four similar experiments. (B) Cell-associated CD317 levels relative to MAPK are given in arbitrary units after quantification of the western blots shown in A. (C) Cells from (A) were processed for microscopic analysis to visualize p24CA+ aggregates (red, upper row), CD4 expression (red, middle row) or HA-CD317 expression (red, bottom row) in transfected, HIV-expressing (GFP-positive) cells. Arrow heads indicate plasma membrane or intracellular p24CA+ aggregates. Scale bar: 10 μm. Shown are representative images of four independent experiments. (D) Quantification of the frequency of cells that displayed p24CA+ aggregates (white bars), co-expressed provirus-encoded GFP (+indicated Vpus) and HA-CD317 (black bars) or co-expressed GFP (+indicated Vpus) and CD4 (grey bars) in cells from the experiment shown in (C). (E-G) In cells from (A) surface levels of stably expressed CD4 and transiently expressed CD317 were monitored by flow cytometry as a function of the provirally expressed GFP and for the indicated co-expressed Vpu proteins. Panel E shows representative FACS dot plots. For quantification, the mean fluorescence intensity (MFI) for surface-exposed CD4 and CD317 was determined on highly GFP-positive cells in the R3 gate relative to the MFI of GFP-negative cells in the R2 gate (see panel (E) for gating and MFI values). MFI values obtained for control cells, not expressing Vpu, were set to 100% (panels F, G). Shown are arithmetic means ± SEM from 3 to 4 independent experiments. Student's t-test: **p < 0.01.