Fig. 3
From: Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells
HIV-1 restriction and dNTP levels in U937 cells expressing SAMHD1 T592 mutants (A and B) HIV-1 restriction assay in (A) differentiated and (B) cycling U937 cells. Cells were first transduced with a bicistronic construct expressing WT SAMHD1 or the indicated mutant and YFP, followed by infection with HIV-GFP. Cells were harvested after 72 h and analysed by flow cytometry. The infectivity ratio was calculated by dividing the percentage of GFP positive cells in the YFP (SAMHD1) positive population by the percentage of GFP positive cells in the YFP (SAMHD1) negative population. The bars show the average of three biological repeats and error bars represent the SD. The T592 mutants, phosphomimetic mutants, and inactive negative controls are shown in blue, grey, and black, respectively. C and D Measurements of dNTP levels from (C) differentiated and (D) cycling U937 cells expressing SAMHD1 and indicated mutants (Neg = untransduced U937 cells). The amount of dATP, TTP, dCTP, and dGTP within the total bar stack are shown in blue, yellow, beige, and red. The values are an average of two technical repeats and error bars represent the SEM. E The effect of HU treatment on the levels of dNTPs. Cycling parental U937 (solid circles), or WT SAMHD1 transduced, U937SAMHD1 (open circles) were treated with 100 µM (red line) or 1 mM (orange and blue lines) HU or untreated (grey and black lines) and samples were taken for dNTP analysis at time points up to 72 h after addition of HU. Dashed and dotted grey thresholds indicate the dNTP levels measured for untreated differentiated U937 and U937SAMHD1 cells respectively. Each point represents the average sum of all four nucleotides (pmole per 106 cells) from two technical repeats. F HIV-1 Restriction assay in cycling U937 cells transduced with WT SAMHD1 (red bars), T592V or T592C mutants (blue bars) or catalytically inactive HD206-7AA and R164A controls (black bars) following 1 mM HU treatment for 72 h (dotted bars) or untreated (solid bars). Restriction assay was performed as above. The bars are the average of three biological repeats and error bars represent the SD