Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection

Abstract

SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.

FIGURE 1.

SAMHD1 mutations located outside the catalytic domain modulate its ability to inhibit HIV-1 infection. A, schematic representation of the human SAMHD1 protein (626 residues). The locations of the SAM domain, allosteric dGTP effector binding site (dGTP), the HD/COG1078 phosphohydrolase domain (HD), and the divergent C terminus (C-term), which is targeted by SIVmac/HIV-2 Vpx, are shown. B, expression of SAMHD1 protein variants in U937 cells. Aliquots of cell extracts used for the luciferase assays shown in C were immunoblotted for SAMHD1 via its N-terminal epitope tag, and α-tubulin (α-Tub), as a loading control. U937 cells transduced with an empty MSCV puro vector (M) were used as a negative control. Fluorescent signals were quantified with an Odyssey infrared imaging system. C, effect of SAMHD1 mutations on its ability to inhibit HIV-1 infection. Wild type and mutant SAMHD1 proteins were expressed in U937 cells by retroviral transduction over a range of multiplicities of infection in dose-response experiments. The transduced cells were induced to differentiate toward macrophages and then challenged with a single cycle vesicular stomatitis virus glycoprotein pseudotyped HIV-1 carrying a luciferase reporter gene, in the absence (black bars) or presence (gray bars) of SIV virus-like particles loaded with Vpx (4, 12) in triplicate wells. Luciferase activity in cell extracts was quantified 2 days later and is shown in arbitrary units, with ± 1 σ error bars. D, mutations outside the HD domain modulate SAMHD1 potency. The scatter plots show luciferase reporter activity (from C) as a function of SAMHD1 variant protein expression level (B) normalized to that of the wild type protein.

FIGURE 2.

In vitro dNTPase activity of recombinant SAMHD1 proteins. A, 150-ng aliquots of SAMHD1 protein variants were separated on Novex 4–12% Bis-Tris gradient gels along with serial 2-fold dilutions of full-length SAMHD1 standards (1–626 (2x); 300, 150, and 75 ng) and revealed by silver staining. B, mutations do not alter SAMHD1 preference for dNTP substrates. Reactions containing wild type (1–626) or the indicated mutant SAMHD1 proteins and 100 μm each dATP, dGTP, dCTP, and TTP were incubated in the presence of 5 mm MgCl₂ for 20 min and deoxyribonucleoside (dN) products of the reactions quantified by HPLC. The average amounts of dA, dG, dT, and dC generated in each reaction, from triplicate samples in two experiments ± standard error, expressed as deoxyribonucleoside/SAMHD1 (mol/mol), are shown. C, dGTP-dependent dATPase activity of wild type and mutant SAMHD1 proteins. SAMHD1 proteins were incubated with dGTP, dATP, dCTP, and TTP for the indicated times, and the amount of dA generated at each indicated time is shown. The standard error from triplicate samples was <5% and usually <2% of the values shown.

FIGURE 3.

SAMHD1 C terminus is required for efficient depletion of dATP in vivo. A, in vivo activity of mutant SAMHD1 proteins. U937 cells expressing SAMHD1 wild type or variants over a range of levels were induced to differentiate toward macrophages, and dATP pools were determined by LC-MS 3 days later. The average dATP level/10⁶ cells from triplicate samples in three representative experiments ± standard error is shown for each SAMHD1 variant. α-Tub, α-tubulin. The vertical line in the middle panel indicates the place where the two parts of this blot are joined. B, SAMHD1 levels in U937 cells. Aliquots of extracts from SAMHD1-expressing U937 cells used for the dATP determinations shown in A were immunoblotted for SAMHD1, via its N-terminal epitope tag, and α-tubulin, as a loading control. Fluorescent signals were quantified using an Odyssey infrared imaging system. M, control U937 cells transduced with empty MSCV vector. C, in vivo potency of mutant SAMHD1 proteins. For each SAMHD1 variant, the amount of dATP, expressed as pmol/10⁶ cells (from A), is plotted as a function of SAMHD1 expression level (from B), normalized to that of wild type SAMHD1.

FIGURE 4.

dGTP induces SAMHD1 tetramerization. A, characterization of quaternary states of recombinant SAMHD1 by SEC-MALS. SAMHD1 (25 μm) alone (red trace) was injected into an analytical gel filtration column at a flow rate of 0.5 ml/min, and the absorbance trace was recorded at 280 nm. The absorbance (right y axis) and MALS molecular mass profile (left y axis) across the elution volume are shown. The average molecular mass of species eluting between 12.6 and 14.2 ml was estimated to be 110 kDa, by MALS. Of note, the calculated molecular mass of SAMHD1 is 75 kDa. Mixtures of SAMHD1 with two different concentrations of dGTP (30 μm, green trace; and 60 μm, blue trace) were also analyzed by SEC-MALS. The elution volumes for tetrameric SAMHD1 species (300 kDa) and a mixture of dimeric and monomeric protein species (110–120 kDa), estimated by MALS, are indicated by arrows. B, SDS-PAGE profiles of chemically cross-linked dGTP-induced SAMHD1 oligomers. Recombinant wild type and variant SAMHD1 proteins with mutations in the predicted dGTP-binding allosteric site (D137A) or catalytic center (HD206RN) (17) were preincubated with dGTP in a reaction buffer, as indicated, chemically cross-linked, and separated on 4–12% Novex gels, and the proteins were visualized by silver staining. The positions of protein bands corresponding to SAMHD1 oligomers and molecular masses of proteins in HiMark protein standards (M) are indicated.

FIGURE 5.

SAMHD1 oligomers in intact cells. A, SAMHD1 is oligomeric in intact U937 cells. U937 cells expressing epitope-tagged hfa-SAMHD1 were cross-linked (C) with formaldehyde at the indicate concentrations (FA%). Aliquots of cell extracts supplemented with gel loading buffer were separated by SDS-PAGE and blotted with FLAG-M2 antibody reacting with the FLAG tag at the N terminus of SAMHD1. A subset of samples was heated at 90 °C for 20 min to partially reverse the cross-links (R) prior to separation and blotting. Bands corresponding to monomeric and slower migrating cross-linked forms of SAMHD1 are indicated. B, characterization of cross-linked SAMHD1 forms purified from U937 cells. hfa-tagged SAMHD1 was purified from U937 cells cross-linked with 0.1 or 0.2% formaldehyde, by two sequential immunoprecipitations via HA and FLAG tags. Cross-links were partially reversed (R; lanes 5 and 6) or not (C; lanes 2–4), and the proteins were resolved as described for A above. A mock immunoprecipitate from U937 cells not expressing SAMHD1 was used as a negative control (lane 2). Recombinant SAMHD1 protein cross-linked with formaldehyde in the presence (lanes 7 and 8) or absence (lane 9) of dGTP was resolved in parallel, as control. Molecular masses of proteins in Mark12 protein standards (M) are indicated. Protein bands were revealed by silver staining.

FIGURE 6.

Tetrameric SAMHD1 is catalytically active dNTPase. A and D, recombinant SAMHD1 (10 μm) was incubated with 30 μm (A) or 60 μm (D) dGTP and separated by size exclusion chromatography at a flow rate of 0.8 ml/min. Elution profiles (A_{280 nm}) with positions of SAMHD1 tetramer and dimer/monomer peaks indicated are shown. Pooled fractions corresponding to the tetramer (positions 1 and 3; elution volumes 10.5–11.0 ml) and dimer/monomer mixture (positions 2 and 4; elution volumes 13.25–13.75 ml) peaks were pooled and assayed for dNTPase activity, shown in B and E and in C and F, respectively. B, C, E, and F, equal volume aliquots (300 μl) of pooled SAMHD1 tetramer or monomer peak fractions were incubated with 100 μm each dATP, dCTP, and TTP in the reaction buffer for the indicated times, and deoxyribonucleoside (dN) products of the reactions quantified by HPLC (C and F). Of note, dGTP was not added to dNTPase assay mixtures. Chromatograms showing separation of the reaction products, collected at the 20-min time point, are shown in B and E. The results of experiments performed with SAMHD1 oligomers formed in the presence of 30 μm dGTP and 60 μm dGTP are shown in B and C and in E and F, respectively.

FIGURE 7.

SAMHD1 C terminus is important for SAMHD1 tetrameric state. A, wild type and the indicated mutant recombinant SAMHD1 proteins were incubated with or without dGTP, chemically cross-linked, separated by SDS-PAGE on 4–12% Novex gels, and visualized by silver staining. B, wild type and mutant SAMHD1 proteins (250 nm) were preincubated with dGTP (200 μm), and the mixtures were separated by size exclusion chromatography, as described under “Experimental Procedures.” The elution profiles were recorded by monitoring fluorescence trace (mV, excitation at 282 nm and emission at 313 nm). Peak elution volumes for SAMHD1 tetramer (tet) and dimer/monomer mixture (di/mono) are indicated (ml), as is the peak corresponding to dGTP, indicated by asterisks. C, SAMHD1 regions flanking the catalytic HD domain influence dGTP-induced tetramer. Wild type and mutant recombinant SAMHD1 proteins (250 nm) were preincubated with dGTP at various concentrations (50–200 μm), and the mixtures were injected into an analytical gel filtration column at a flow rate of 0.8 ml/min. The elution profiles (fluorescence trace, excitation at 282 nm and emission at 313 nm) are shown with peak positions corresponding to tetramer and monomer of SAMHD1 indicated. The tetramer peak height (ordinate) is plotted as a function of dGTP concentration (abscissa).