Figure 5: HIV-1 HA/His6-Vpr functional testing.
From: HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome
(a) Replication curves of HIV-1 HA/His6-Vpr and WT HIV-1 after infection of SupT1 with 5–160 ng p24. Infection is measured by HSA expression, at several days post-infection (p.i.). WT replication is presented as solid lines; HA/His6-Vpr as dashed lines. (b) Increased viral input infection experiment in SupT1, using 160 ng p24 HIV-1 HA/His6-Vpr to monitor induction of cell death. Upper dot plots show forward (FSC) and side scatter (SSC), lower plots show HSA staining versus SSC for cells gated in the live scatter gate shown in the upper dot plots. Figures indicate percentage of cells staining positively with the APC labelled antibody. (c) SupT1 infection with VSV-G pseudotyped HA/His6-Vpr virus, 160 ng p24 virus. Upper dot plots show forward (FSC) and side scatter (SSC), lower plots show HSA staining versus SSC for cells gated in the live scatter gate shown in the upper dot plots. Figures indicate percentage of cells staining positively with the APC labelled antibody, gated on live cells as shown. (d) Cell cycle analysis on pNL4-3-transfected 293T cells 48 h post-transfection. 293T cells were transfected with the proviral constructs or Mock transfected, as indicated. Data were analyzed with the FlowJo cell cycle analysis platform on flow cytometry data of DNA staining, applying the Dean-Jett-Fox model. Left panel: histograms from a representative experiment. Separate green curves in the shaded histogram illustrate the fraction of cells in G1, S and G2 phase of the cell cycle. The percentage of cells in G2 phase (represented by green curve covering highest DNA content) is shown for each sample. Right panel: percentage of cells in G2 phase induced by the indicated constructs or Mock transfection. Error bars indicate standard deviation. Mann-Whitney U test comparing samples. N = 8 (Mock), N = 6 (HIV WT/HIV ΔVpr) or N = 5 (HIV HA/Flag-His6-Vpr). Mock-HIV WT p = 0.0007, Mock-HIV ΔVpr p = 0.0062, HIV WT–HIV ΔVpr p = 0.0303, HIV ΔVpr-HIV HA/FLAG-Vpr p = 0.0159, HIV ΔVpr-HIV HA/His6-Vpr p = 0.0317.
