Fig. 3

Distribution of HIV-1 R3A WT or the PTAP^–YP^– and PTAP^– mutants in infected monocyte-derived macrophages (MDMs). Seven-day-old MDMs were infected with HIV-1 R3A WT or the rescued release-defective viruses HIV-1 R3A PTAP⁻YP⁻ or PTAP⁻ (see Additional file 2: Figure S2). Cultures were incubated for a further 7 days, fixed and immunolabelled with antibodies against p24/55 (Kal-1, green), p17 (4C9, red) and CD44 (magenta), and examined by confocal microscopy. a–e Selected single optical sections from z-stacks of MDMs infected with WT (a) or the PTAP^–YP^– mutant (b–e, two different sections are shown for the cell in d). Arrows point out the intracellular plasma membrane-connected compartments (IPMCs). The boxed area in (d) is enlarged in the insets, revealing a single immature virus bud at the cell surface. Scale bars, 20 μm. f Quantitative analysis of virus distributions on MDMs from five different blood donors. Coverslips were scanned systematically and virus-expressing cells scored into three categories of cells where virus was seen in the IPMCs only (blue), in IPMCs as well as the cell surface (grey), or only at the cell surface (white; these latter MDMs lacked CD44⁺ IPMCs). g For selected cells with Gag p24/55 labelling both at the IPMC and the cell surface, the proportion of p24/p55 fluorescence in the IPMC was estimated and plotted. p24/p55Gag fluorescence was primarily in the IPMC for the WT (>80 %), and for cells from two donors infected with PTAP⁻YP⁻ mutant virus (>90 %). See also Additional file 3: Figure S3 and Additional file 4: Figure S4