Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels

Abstract

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.

Keywords: DNA Polymerase; DNA Replication; HIV-1; Macrophages; Nucleoside Nucleotide Analogs.

FIGURE 1.

Knockdown of SAMHD1 decreases NRTI efficacy in THP1 cells. A, Western blot of SAMHD1 expression in differentiated THP1 cells with a scramble shRNA or a SAMHD1-specific shRNA. B and C, THP1 cell lines were differentiated, treated with increasing concentrations of AZT (B) or the NNRTI nevirapine (C), and transduced with HIV-1 D3. To determine transduction efficiency, cells were collected 48 hpt to measure GFP expression by flow cytometry (error bars represent the S.E., n = 3).

FIGURE 2.

Vpx-mediated degradation of SAMHD1 decreases NRTI efficacy in macrophages. A, SAMHD1 expression level in three donors of macrophages (M1, M2, and M3) treated without VLPs or with VLPs ±Vpx for 24 h. B–E, macrophages were treated with VLPs for 24 h, treated with increasing concentrations of AZT (B), ABC (C), ddC (D), or TDF (E), and then transduced with HIV-1 D3. To determine transduction efficiency, cells were collected 7 dpt to measure GFP expression by flow cytometry (error bars represent the S.E., n = 3).

FIGURE 3.

Vpx-mediated degradation of SAMHD1 decreases the efficacy of combination NRTI treatment. A, purified HIV-1 RT protein was used to extend the indicated 5′ ³²P-labeled primer/template with increasing dNTP concentrations (10 nm, 25 nm, 50 nm, 100 nm, 250 nm, 500 nm, and 1 μm) with or without a fixed concentration of AZT-TP and ddCTP. The dNTP concentrations found in macrophages and activated T cells are marked as M and T, respectively. The negative control (−) was without drug or dNTPs, and the positive control (+) was with drug and 1 nm dNTPs. Asterisks in blue and red indicate positions of chain termination for AZT-TP and ddCTP, respectively. F: fully extended 38-mer product; P: unextended 17-mer primer. B, VLP-treated macrophages were treated with 150 nm AZT/400 nm ABC or 150 nm ddC/200 nm TDF and transduced with HIV-1 D3. To determine transduction efficiency, cells were collected to measure GFP expression by flow cytometry (error bars represent the S.E., n = 3).

FIGURE 4.

Vpx-mediated degradation of SAMHD1 decreases NRTI efficacy in activated T cells. A, SAMHD1 expression level in three donors of activated CD4⁺ T cells (T1, T2, and T3) treated without VLPs or with VLPs ±Vpx for 24 h. B, using the same three donors, dNTPs were collected and measured using a single nucleotide RT primer extension assay (10). -Fold increase of dNTPs in VLP +Vpx-treated cells when compared with VLP −Vpx-treated cells is shown (error bars represent the S.E., n = 2). C–F, activated CD4⁺ T cells were treated with VLPs ±Vpx for 24 h, treated with increasing concentrations of AZT (C), ABC (D), ddC (E), or TDF (F), and then transduced with HIV-1 D3. To determine transduction efficiency, cells were collected to measure GFP expression by flow cytometry (error bars represent the S.E., n = 3).

FIGURE 5.

SAMHD1 enzymatic activity toward ddNTPs and allosteric activation with ddGTP. A, recombinant SAMHD1 and a standard curve of bovine serum albumin (8 μg-0.5 μg, BSA) were run on SDS-PAGE and stained with Coomassie Blue. MW Std., molecular weight standard. B–E, purified SAMHD1 was incubated with the indicated nucleotide in the presence or absence of 100 μm dGTP as an allosteric activator. Nucleotide substrates remaining in the triphosphate form were separated and quantified using HPLC (error bars represent the S.E., n = 3). F, SAMHD1 was incubated with 1 mm dATP and 500 μm of dGTP or ddGTP. The quantity of dATP remaining after incubation was measured using HPLC and quantified as described under “Experimental Procedures” (p, 0.2599). Statistical significance was measured using an unpaired t test with a Welch's correction, n = 3.