Fig. 4

SAMHD1 restriction in U937 cells infected with HIV-1 RT V148I and Q151N mutants Restriction of A and B HIV-1 RT-V148I and C and D RT-Q151N RT VLPs in A and C differentiated and B and D cycling U937 cells expressing the indicated SAMHD1 T592 mutant. 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. E Restriction assay of HIV-1 RT-Q151N 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 show the average of at least three biological repeats and error bars represent the SD. *The low abundance of double positive cells (GFP⁺YFP⁺  < 0.05%) for WT SAMHD1, T592V, and T592C HU-treated cells prevented determination of a reliable infectivity ratio