Functional analysis of Vpu mutated at positions that formed efficient crosslinks with Tetherin.

<p>(<b>A</b>) 293T cells were cotransfected with HIV-1 proviral (pNL4-3) plasmids expressing various mutant Vpu proteins, and with increasing amounts of a plasmid encoding WT Tetherin-HA. Infectious virion yield was measured using TZM indicator cells and is given in relative light units (RLU). (<b>B</b>) Western blot analysis of cell lysates and released particles from samples in panel (<b>A</b>) that were cotransfected with 0, 25, or 100 ng of Tetherin-HA. Blots of cell lysates were probed with α-HA and capsid α-CA antibodies. Blots of virion lysates were probed with α-CA antibodies. (<b>C</b>) Western blot analysis of Vpu expression from samples in panel (<b>A</b>) that were transfected with 0 ng of Tetherin. Blots were probed with an α-Vpu antibody. (<b>D</b>) 293T cells stably expressing Tetherin-HA were transduced with VSV-G pseudotyped HIV-1 based vector encoding GFP and various mutant Vpu proteins. Cells transduced at an MOI of 0.5 were surface-stained using an α-Tetherin antibody and analyzed by FACS to determine the relative level of Tetherin-HA expression. The FACS plots show examples of this assay. (<b>E</b>) Cells transduced with the same vectors used in (<b>D</b>), but at an MOI of 3, were lysed and analyzed using quantitative fluorescence-based Western blotting. Blots were probed with antibodies specific for Vpu, GFP, tubulin, and HA, as indicated. (<b>F</b>) Chart summarizing data from assays carried out as described in (<b>D</b>) and (<b>E</b>). For FACS analysis, the geometric mean fluorescence after gating on GFP-positive cells was quantified (gray bars). For Western blot analysis band intensities were quantified using a LI-COR scanner (red bars). The mean and standard deviation of the relative amounts of Vpu expression (from four independent experiments) is plotted, with the amount of Tetherin expression observed following transduction with the ΔVpu vector set to 100%.