Fig 2: Analysis of function of p53 and its downstream gene p21 during HIV infection in hMDMs. a and b Analysis of p53, p-p53 (S15) and p21 proteins in hMDMs after HIV-1 infection by Western blot. 3.0 MOI VSV-G pseudotyped HIV-1 GFP+ virus was used to infect 0.5 × 106 hMDMs in a 24 well plate, Results from two different donors were shown. c Western blot analysis of SAMHD1, pSAMHD1 (T592) and RNR2 after siRNA knockdown of p53 and siRNA knockdown of p21 in hMDMs. d Percentages of p21 protein and p53 protein after siRNA knockdown. The non-targeting (NT) siRNA was used as negative control. e Quantification of HIV-1 Luc+ infection after siRNA knockdown of p21 and siRNA knockdown of p53 in hMDMs. f Analysis of SAMHD1 and pSAMHD1(T592) in HIV-2 infection after siRNA knockdown of p21 in hMDMs. g The percentage of p21 after siRNA knockdown in hMDMs. h Quantification of HIV-2 infection in hMDMs transfected with p21 siRNA and non-targeting siRNA. The non-targeting (NT) siRNA was used as negative control. Luciferase assay was performed at 48 h after HIV-2 Luc+ infection. The results represented a triplicate experiment. In Student’s t-test, p value < 0.05 is indicated by *

Fig 3: Productive infection of iDCs by cell-to-cell fusion with infected T cells escapes SAMHD1 restriction. (A) NLAD8-infected Jurkat cells were cocultured for 24 h with iDCs derived from 4 different donors with or without AZT. T cells were then eliminated by extensive washes as described previously, and iDCs were cultured with or without AZT for different period of time. Cell culture supernatants from iDCs were collected after 3, 6, 9, or 12 days, and viral p24 was quantified. Each experiment was performed in duplicate. NI, noninfected Jurkat cells were cocultured with iDCs. (B) NLAD8-infected Jurkat cells were cocultured for 6, 24, or 72 h with iDCs with or without AZT, and the percentage of Gag+/CD11c+ iDCs was then evaluated by flow cytometry. The results represent means of 3 independent experiments performed with iDCs of 3 different donors. Error bars represent 1 SEM. Statistical significance was determined using the Mann-Whitney U-test (ns, P > 0.05; **, P < 0.01). (C) iDCs were transfected with siRNA targeting SAMHD1 (siSAMHD1) or scrambled siRNA (siCtrl) used as a control. Lysates from transfected cells were analyzed by Western blotting using anti-SAMHD1 and anti-β-actin antibodies. The band intensities were quantified using NIH Image software, and the signal measured in siRNA-transfected cells was normalized to the signal obtained for actin. The indicated values are expressed as percentages of the signal intensity relative to siCtrl (100%) and are representative of 3 independent experiments. (D to F) iDCs transfected with siCtrl (blue bars) or siSAMHD1 (hatched bars) were infected by cell-free NLAD8 viruses produced by Jurkat cells (CF) or by coculture with NLAD8-infected Jurkat cells (CTC) for 24 h. (D) The percentage of Gag+/DC-SIGN+ iDCs was evaluated by flow cytometry directly after 24 h of infection with CF or CTC. The results are expressed as the percentage of Gag+ iDCs relative to that obtained with siCtrl-transfected iDCs (100%). (E and F) Quantification of p24 released in cell culture supernatants from siRNA-transfected iDCs infected by CF or CTC for 24 h and then cultured for 6 more days. The results are expressed as the percentage of p24 released relative to that released by CTC infection (E), or as the percentage of p24 released relative to that released by siCtrl-transfected iDCs (F). Results represent means of 4 independent experiments performed with iDCs from 4 different donors. Error bars represent 1 SEM. Statistical significance was determined using the Mann-Whitney U-test (ns, P > 0.05; *, P < 0.05; **, P < 0.01). (G) MDMs were infected by cell-free NLAD8 viruses produced by CD4+ primary T cells (Cell-free, left panels) or by coculture with NLAD8-infected primary T cells (Cell-to-cell, right panels) for 24 h. Cell lysates were then analyzed by Western blotting using anti-SAMHD1, anti-phospho-SAMHD1, anti-HIV-1 p24, and anti-β-actin antibodies.

Fig 4: SAMHD1 (but not DCK) levels determine sensitivity to CNDAC and inversely correlate with CNDAC-triphosphate (CNDAC-TP) in leukaemia cell lines. (A) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMHD1), and DCK in 13 AML cell lines. GAPDH served as loading control. Uncropped Western blots are presented in Supplementary Figure 1. (B) CNDAC concentrations that reduce the viability of AML cell lines by 50% (IC50). Horizontal lines and error bars represent means ± SD of three independent experiments. (C) CNDAC triphosphate (CNDAC-TP) levels determined by LC–MS/MS. Horizontal lines and error bars show means ± SD of three independent experiments. (D, E) Correlation of the CNDAC IC50 values with cellular DCK (D) or SAMHD1 (E) protein levels, quantified using near-infrared Western blot images to determine the ratio DCK/ GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (F, G) Correlation of CNDAC-TP levels with cellular DCK (F) or SAMHD1 (G) protein levels in AML cell lines, quantified using near-infrared Western blot images to determine the ratio DCK/GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (H) Analysis of SAMHD1 promoter methylation in AML cell lines through amplification of a single PCR product (993-bp) corresponding to the promoter sequence after HpaII digestion. A 0.25-kb fragment of the GAPDH gene lacking HpaII sites was PCR-amplified using the same template DNA served as loading control. THP-1 served as control cell for an unmethylated SAMHD1 promotor, while JURKAT served as control cell for a methylated promotor. (I) Correlation of CNDAC IC50 values in 26 ALL cell lines (11 T-ALL, 15 B-ALL) with SAMHD1 protein levels, quantified using near-infrared Western blot images to determine the ratio SAMHD1/ GAPDH relative to the positive control THP-1. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (J-L) Comparison of SAMHD1 protein levels (J), CNDAC IC50 values (K) and CNDAC-TP levels determined by LC-MS/MS (L) in T-ALL and B-ALL cells. Each point represents the mean of three independent experiments. One-tailed Student’s t-tests were used to compare means in T-ALL and B-ALL cells (represented as horizontal lines ± SEM)

Fig 5: SAMHD1 knockdown elicits influenza A replication and abolishes CCL5 inhibitory effect on viral replication. A549 cells were transfected with siRNA for SAMHD1 or its scramble sequence at a concentration of 100 nM in Opti-MEM, using Lipofectamine 2000. At 6 hours post transfection, A549 cells were exposed or not to CCL5 (15 ng/mL) or IFN-α (10 ng/mL). After 18 h of exposure (A, B), monolayers were lysed with RIPA buffer for western blot assay to SAMHD1 and β-actin, as a housekeeping protein, or (C, D) infected with influenza A virus at MOI 2. At 24 hpi, supernatants were harvested for extraction of viral RNA and the viral titer measured through qRT-PCR (copies/mL) and by TCID50 assays. (E) Supernatants of those cultures were also quantified for LDH activity, as a marker of cell death. Data were obtained from 3 independent experiments with technical duplicates. *p < 0.05, **p < 0.01 and ***p < 0.001.