Fig. 1

Characterisation of HIV-1 R3A p6 domain mutants in HEK 293 T cells. HEK 293 T cells were transfected with the indicated HIV-1 pNL4.3-R3A constructs (see Additional file 2: Figure S2A), and cultured for 24 h before analysis. a Western blot of cell lysates and released virions purified from the media. Blots were probed with antibodies against viral p24/55 or adaptin-γ as a loading control. The positions of p55Gag, Gag protein lacking the p6 domain (p55Δp6), the p41 cleavage product and mature p24 are indicated. Band signal intensities were quantified using ImageJ to determine the virus release efficiencies shown below the blot (data from two independent experiments ± standard deviation). b Immunofluorescence staining of semi-thin cryo-sections (0.5 μm) with antibodies against p24/55 (38:96 K and EF7, green) and p17 (4C9, red). Bright puncta of p24/p55 (e.g. at the green arrowheads) indicate assembling viruses. For the R3A WT, some mature cell surface puncta were labelled for p17 (red arrowheads) suggesting they are mature virions, but for the PTAP^– and Δp6 mutants, the cell surface contained only p24/p55-labelled puncta (green) devoid of p17 staining, indicative of immature virus buds. Scale bars, 10 μm. c Ultrastructure of HEK 293 T cells expressing the indicated HIV-1 R3A constructs. Transfected cells were embedded in Epon and processed for electron microscopy. Cells expressing the HIV-1 R3A WT contained rare mature virus particles such as the group of particles trapped between adjacent cells shown here. Arrows mark electron-dense cores visible in some of the particles. In contrast, only immature budding-arrested virus particles were seen on cells expressing the PTAP^–, PTAP^–YP^– and Δp6 mutant proviruses. Scale bars, 200 nm