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. 2012 Mar 29;8(3):e1002609. doi: 10.1371/journal.ppat.1002609

Figure 5. The second alpha helix of Vpu can be functionally replaced by the D/EXXXLL endocytic signal from HIV-1 Nef.

Figure 5

(A) Schematic representation of Vpu ENTSLL and Vpu-Nef chimeric constructs. (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. (C) Infectivity of viral supernatants from B was determined on HeLa-TZMbl cells as in Figure 1B. Error bars represent standard deviation of three independent experiments. (D) 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 Figure 2D. (E) 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.