SAMHD1 has differential impact on the efficacies of HIV nucleoside reverse transcriptase inhibitors

Abstract

Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.

FIG 1

SAMHD1 does not efficiently hydrolyze NRTI-TPs. SAMHD1 (5 μM) was incubated at 37°C with NRTI-TP or dNTP (500 μM), in the presence of dGTP (100 μM) and MgCl₂ (10 mM). Reactions proceeded for 3, 6, or 20 h for NRTI-TPs and for 5, 15, or 30 min for dNTPs. Reactions were terminated by 10-fold dilution into 25 mM Tris (pH 8.0)-12.5% acetonitrile, and mixtures were analyzed by anion-exchange HPLC (DNAPac PA100 column). (A) Representative chromatograms for dATP hydrolysis. dG, deoxyribosylguanine. (B and C) Data from at least duplicate experiments for dNTP (B) or NRTI-TP (C) hydrolysis, plotted as percent hydrolysis over time (with GraphPad Prism 5). ddATP, dideoxyadenosine triphosphate; ddGTP, dideoxyguanosine triphosphate. (D) Chromatogram for dATP hydrolysis after 30 min of incubation with the SAMHD1 hydrolase active-site D207A mutant, dGTP, and MgCl₂.

FIG 2

Knockdown of SAMHD1 has differential effects on NRTI activation. PMA-differentiated parental and THP-1_KD-SAMHD1 cells were treated with 1.5 μM (0.5 μCi) [2-¹⁴C]AZT or [4-¹⁴C]d4T. After 24 h at 37°C, the cells were lysed, the dNTP and NRTI metabolites were separated by anion-exchange HPLC (DNAPac PA100 column), and the NRTI metabolites in the collected fractions were quantified with a liquid scintillation counter. Data represent the mean ± standard deviation (SD) from 2 independent experiments. *, P < 0.05.

FIG 3

Exogenously added dT, but not dC, affects AZT and d4T potencies. TZM-bl cells were treated with PBS, 100 μM dT, or 100 μM dC or 100 µM dA (as controls for noncompeting nucleosides) and infected with HIV-1_NL4-3 at an MOI of 0.02, in the presence of increasing concentrations of inhibitor (AZT or d4T). At 48 h postinfection, cells were lysed and luciferase activity was detected. Luciferase activity at various drug concentrations was plotted using the one-site competition equation in GraphPad Prism 5, and data were normalized to the no-nucleoside control results. Data represent the mean ± SD from at least three independent experiments. Data in the table represent the mean ± SD from at least three independent experiments. Shown also are the fold changes in the EC₅₀ of NRTI in the presence or absence of cognate nucleoside, which indicate change in sensitivity to AZT/d4T, 3TC, or ddI, in the presence of dT, dC, or dA, respectively. ND, not determined.