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. Author manuscript; available in PMC: 2017 Aug 1.
Published in final edited form as: Virology. 2016 May 14;495:92–100. doi: 10.1016/j.virol.2016.05.002

Fig 4. Knockout of SAMHD1 increases HIV-1 infection of non-differentiated and differentiated THP-1 cells.

Fig 4

(A) THP-1 SAMHD1 control and KO cells were grown in the absence or presence of 100 ng/ml of PMA for 24 hours to induce differentiation into macrophage-like cells. Immunoblotting analysis using a specific SAMHD1 antibody confirmed efficient knockout of SAMHD1 in KO cells. (B) HIV-1 infection is increased in KO cells compared to control cells. Control and SAMHD1 KO cells, differentiated or not with PMA as described in (A), were infected with a single-cycle HIV-1-Luc/VSV-G at a multiplicity of infection (MOI) of 1. At 24 and 48 hours post-infection, HIV-1 infection efficiency was measured by luciferase assay. Luciferase values were normalized for protein concentration. The infection level in the control cells at 24 hours post-infection was set as 1 and relative values are shown. A representative experiment performed with four replicates per sample is presented. Statistical analysis was performed with the unpaired T-test with Welch’s correction (**P=0.0019, ***P<0.0001). (C) SAMHD1 overexpression in KO cells. KO cells were transduced with LVX-IRES-mCherry control lentiviral vector (Lvx) or LVX-SAMHD1-IRES-mCherry vector (SAM). Cells were sorted by flow cytometry and SAMHD1 expression was analyzed in six Lvx and four SAM clones after differentiation with PMA by immunoblotting using a SAMHD1 specific antibody. GAPDH was used as loading control. (D) Rescue of SAMHD1 in KO cells restores SAMHD1-mediated depletion of dNTP levels. Intracellular dNTP concentration was measured in differentiated cells as described in Fig. 1D. (E) Effect of SAMHD1 KO and overexpression on HIV-1 restriction. Cells were infected with a GFP-expressing HIV-1 virus and, after 72 hours, the percentage of GFP-positive cells was measured by flow cytometry. While silencing of SAMHD1 results in more efficient HIV-1 infection, overexpression of SAMHD1 in KO cells restores SAMHD1 restriction effects. Counteraction of SAMHD1-mediated viral restriction was detected in SAM cells co-treated with Vpx and dNs.