SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates

Abstract

SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

Figure 1

SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated THP-1 cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ-³²P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ-³²P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).

Figure 2

Vpx increases the intracellular pool of dNTP in MDM. (a) MDM (2 × 10⁶) from three healthy donors were pre-incubated for 4 h with Vpx-containing or control VLP or with 1.5 mM dN. The cells were infected with HIV-GFP reporter virus (MOI=1) and the GFP⁺ cells were quantified by flow cytometry. (b) MDM were treated with VLP, dN or 2 mM hydroxyurea. dNTP were visualized by the single nucleotide extension method. (c) The data were quantified and are plotted as a histogram. The K_m and K_d values of HIV-1 RT for dNTP substrates are indicated.

Figure 3

SAMHD1 reduces the intracellular dNTP pool and is counteracted by human and rhesus but not mouse SAMHD1. U937 cells were transduced with a control lentiviral vector (pLenti), or with a vector that expressed human SAMHD1 (hu high and hu mod), human HD206-7AA SAMHD1 (hu HD/AA), rhesus macaque SAMHD1(rh) or the two mouse SAMHD1 isoforms (mu iso1 and mu iso2). Two independent control cell lines were established (pLenti#1 and pLenti#2) in parallel with human SAMHD1 cell lines that expressed at high or moderate levels (hu high and hu mod). (a) The cells were either untreated (left) or PMA-treated (right) and SAMHD1 expression level was determined by immunoblot analysis. (b) dNTP levels were quantified in the untreated and PMA-treated U937 cells. (c) The PMA-treated U937 cell lines were infected with Δvpx or wild-type SIVmac239-HSA reporter viruses. The infected cells were stained with anti-murine CD24 monoclonal antibody and the infected cells were quantified by flow cytometry. (d) The cell lines were infected with HIV-1 luciferase reporter virus and luciferase activity was determined after two days.

Figure 4

Vpx rescues the infection of an HIV-1 that encodes a mutant RT with a reduced affinity for dNTP. (a) MDM were pre-incubated with Vpx-containing or control VLP and then infected with wild-type or RT V148I mutant HIV-GFP at an MOI of 1. Four days post-infection, GFP⁺ cells were quantified by flow cytometry. (b) The efficiency of infection in the presence and absence of Vpx is displayed as the ratio of mutant/wild-type virus. A ratio of 1 indicates equivalent infection efficiency of wild-type and RT V148I mutant HIV-1.

Figure 5

The salvage pathway of dNTP synthesis partially rescues infectivity of Δvpx SIVmac in MDM. MDM were pre-incubated with Vpx-containing and control VLP and were infected with Δvpx SIVmac GFP reporter virus (MOI=1). The salvage pathway of dNTP synthesis was stimulated by addition of 0.75, 1.5 or 2.5 mM dN to the media (from 2 h before infection to 20 h post-infection) where indicated. Four days post-infection, the percent GFP⁺ MDM was determined by flow cytometry. Three representative donors are shown.