SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells

Abstract

SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs.

Figure 1. The effects of d4T-TP,ddI-TP,ddA-TP, ABC-TP and CBV-TP on SAMHD1 triphosphohydrolase activity.

(A) Hydrolysis of 0.3 mM TTP, d4T-TP or ddI-TP in the absence (left) and presence (right) of 0.1 mM activator GTP. (B) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, d4T-TP or ddI-TP as activators. (C) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, d4T-TP or ddI-TP. Error bars are the standard error of the mean (SEM) of three independent measurements. (D) Concentration dependence of SAMHD1 ddI-TP hydrolysis in the presence of 0.2 mM GTP (saturating activator concentration). Nonlinear least squares fitting using a Hill equation gives the apparent binding constant K_S = 226 ± 12 μM, catalytic constant k_cat = 0.24 ± 0.04 s⁻¹ and the Hill coefficient n = 1.7 ± 0.1 (Mean ± SEM). (E) ddI-TP allosteric activation of TTP hydrolysis. Rates were determined for 1 mM TTP at varying dd-ITP concentration. Nonlinear least squares fitting gives only lower estimates for the maximal rate and the ddI-TP concentration at half maximal activation of k_max > 0.1 s⁻¹ and K_a > 300 μM. (F) Hydrolysis of 0.3 mM TTP, ABC-TP, CBV-TP and ddATP in the absence (left) and presence (right) of 0.1 mM activator GTP. (G) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, ABC-TP, CBV-TP or ddATP as activators. (H) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, ABC-TP, CBV-TP or ddATP. Error bars are the range of data from two independent measurements.

Figure 2. Anti-HIV-1 activities of ACV, GCV and CFB in monocytoid cell lines.

PMA⁻ (red) and PMA⁺ (blue) U937 (A–C) and THP-1 (D–F) cells were cultured in the presence of increasing concentration of Aciclovir (ACV) (A,D), Ganciclovir (GCV) (B,E) and Clofarabine (CFB) (C,F). Cells were incubated with HIV-1-GFP and the percentage of infected GFP⁺ cells, measured by flow cytometry. To account for differences in overall infectivity of PMA⁻ and PMA⁺ cells, in each panel the data is plotted as the normalised percentage of GFP⁺ cells against drug concentration. Error bars represent the standard deviation from at least two independent experiments.

Figure 3. Anti-HIV-1 activities of nucleoside analogues in U937 cells expressing SAMHD1.

Antiviral activities were determined in undifferentiated (PMA⁻) and differentiated (PMA⁺) U937 cells expressing SAMHD1 or the mutant HD206-7AA. Cells were cultured in the presence of increasing concentration of Aciclovir (ACV) (A), Ganciclovir (GCV) (B), Clofarabine (CFB) (C), Stavudine (d4T) (D), Didanosine (ddI) (E) and Abacavir (ABC) (F). Cells were incubated with HIV-1-GFP and the percentage of infected GFP⁺ cells was measured by flow cytometry. PMA⁻ cells expressing SAMHD1 (red filled circles), PMA⁺ cells expressing the mutant HD206-7AA (blue open circles) and PMA⁺ cells expressing SAMHD1 (blue filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP⁺ cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.

Figure 4. Anti-HIV-1 activities of nucleoside analogues in differentiated THP-1 cells after Vpx knockdown of endogenous SAMHD1 expression.

Antiviral activities were determined in differentiated (PMA⁺) THP-1 cells expressing endogenous SAMHD1 and after transduction with Vpx. Cells were cultured in the presence of increasing concentrations of Aciclovir (ACV) (A), Ganciclovir (GCV) (B), Clofarabine (CFB) (C), Stavudine (d4T) (D), Didanosine (ddI) (E) and Abacavir (ABC) (F). Cells were incubated with HIV-1-GFP and the percentage of infected GFP⁺ cells was measured by flow cytometry. Cells expressing SAMHD1 (Vpx⁻) (blue filled circles), cells transduced with Vpx (Vpx⁺) (red filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP⁺ cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.