The second alpha helix of Vpu can be functionally replaced by the D/EXXXLL endocytic signal from HIV-1 Nef.

<p>(<b>A</b>) Schematic representation of Vpu ENTSLL and Vpu-Nef chimeric constructs. (<b>B</b>) 293T cells were transfected with NL4.3 wt of NL4.3 delVpu proviral plasmids in combination with tetherin and indicated pCR3.1 Vpu-HA or Vpu-Nef-HA chimera. 48 h post transfection, cell lysates and pelleted supernatant virions were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu-HA and Hsp90, and analyzed by LiCor quantitative imager. (<b>C</b>) Infectivity of viral supernatants from <b>B</b> was determined on HeLa-TZMbl cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat-1002609-g001" target="_blank">Figure 1B</a>. Error bars represent standard deviation of three independent experiments. (<b>D</b>) HeLa cells were co-transfected with Vpu or indicated Vpu mutant and a GFP expression construct. Cell surface staining for endogenous tetherin was analyzed by flow cytometry 48 h post transfection, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat-1002609-g002" target="_blank">Figure 2D</a>. (<b>E</b>) HeLa cells were transfected with Vpu-Nef or Vpu-Nef-mu CherryFP fusions (red) and counterstained for TGN46 (green) and DAPI (blue) and examined by confocal microscopy.