Fig. 1

Membrane-proximal, hinge region E28/L33 residues are important for Vpu localization in the TGN. a Schematic representation of the structural domains and the sequence of the prototypical HIV-1 clade B NL4-3 Vpu (NL4-3, WT). Shown also are sequences of various Vpu mutants used in the study. b Intracellular localization of the Vpu mutants. HeLa cells were transfected with proviral plasmids encoding either WT Vpu or one of E28A/L33A, E59K/L63F, E28A/L33A-E59K/L63F and A10L/A14L/A18L Vpu mutants and were co-stained with anti-TGN46 (red, for TGN) and anti-Vpu (green) Abs as well as with DAPI (grey, for nucleus). Shown are representative confocal microscopy pictures for each of the Vpu mutants, with two prototypical patterns (1&2) of localization observed with the E28A/L33A mutant. c, d Quantification of the co-staining of anti-Vpu and anti-TGN46 Abs obtained from at least 50 distinct transfected cells per mutant. Shown are Pearson correlation coefficients (PCC) for each mutant (c) as well as Vpu distribution beyond the TGN (d). The percentage of Vpu distributing beyond the TGN was determined by calculating the ratio of the intensity of Vpu not co-staining with TGN versus total Vpu intensity in transfected cells. The white bars in B represent a distance of 10 µm and the horizontal lines (c, d) represent mean values of the PCC (c) and percentage of Vpu distributing beyond the TGN (d). Statistical analyses were performed using Mann–Whitney test