SAMHD1 is a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and inhibits the ability of retroviruses, notably HIV-1, to infect myeloid cells. Although SAMHD1 is expressed in both cycling and noncycling cells, the antiviral activity of SAMHD1 is limited to noncycling cells. We determined that SAMHD1 is phosphorylated on residue T592 in cycling cells but that this phosphorylation is lost when cells are in a noncycling state. Reverse genetic experiments revealed that SAMHD1 phosphorylated on residue T592 is unable to block retroviral infection, but this modification does not affect the ability of SAMHD1 to decrease cellular dNTP levels. SAMHD1 contains a target motif for cyclin-dependent kinase 1 (cdk1) ( 592 TPQK 595 ), and cdk1 activity is required for SAMHD1 phosphorylation. Collectively, these findings indicate that phosphorylation modulates the ability of SAMHD1 to block retroviral infection without affecting its ability to decrease cellular dNTP levels.

This work explores the molecular basis by which SAMHD1 blocks HIV-1 infection only when expressed in noncycling cells. Our mass-spectrometry findings revealed that SAMHD1 is unphosphorylated at position T592 in noncycling cells. By contrast, SAMHD1 is phosphorylated at position T592 in all the cycling cells studied here. We studied the role of T592 phosphorylation in the ability of SAMHD1 to block retroviral infection by replacing T592 with phosphomimetic or unphosphorytable residues. SAMHD1 variants with a phosphomimetic residue at position 592 revealed that a phosphorylated SAMHD1 is unable to block retroviral infection without affecting the ability of SAMHD1 to decrease the cellular levels of dNTPs or the intrinsic enzymatic dNTPase activity of the protein. We also showed that the ability of phosphorylation to regulate restriction requires the HD domain and the C-terminal residues 583–626 of SAMHD1. Finally, we explored the nature of the kinase that might be phosphorylating SAMHD1 in cycling cells.

Remarkably, cycling and noncycling cells can express SAMHD1; however, SAMHD1’s antiviral activity is only observed in noncycling cells. Several examples illustrate the fact that SAMHD1 is antivirally active in noncycling cells. Human monocytic THP-1 cells that endogenously express SAMHD1 restrict HIV-1 when cells are differentiated to a noncycling state by treatment with phorbol-12-myristate-13-acetate (PMA) (). Similarly, the human monocytic U937 cells stably expressing an exogenous FLAG-tagged SAMHD1 protein only restrict HIV-1 when cells are differentiated with PMA to a noncycling state. In addition, endogenous expression of SAMHD1 by noncycling cells such as macrophages and dendritic cells shows potent restriction of HIV-1 infection. More recently, it was discovered that noncycling resting CD4T cells potently block HIV-1 infection (). By contrast, the cycling human HeLa and 293T cells that endogenously express SAMHD1 did not show activity against HIV-1 even when SAMHD1 was overexpressed.

SAMHD1 comprises the sterile alpha motif (SAM) and histidine-aspartic (HD) domains. The HD domain of SAMHD1 is a dGTP-regulated dNTPase that decreases the cellular levels of dNTPs (). In agreement with this, the sole HD domain is sufficient to potently restrict infection by different viruses (). The HD domain is also necessary for the ability of SAMHD1 to oligomerize and to bind RNA (). The decrease in dNTP levels in myeloid cells correlates with the inability of lentiviruses to undergo reverse transcription. Even though it is known that SAMHD1 blocks lentiviral infection by depleting the pool of dNTPs, the regulation of the antiviral activity of SAMHD1 is not understood.

Efficient infection of human primary macrophages, dendritic cells, and resting CD4T cells by macaque simian immunodeficiency virus (SIV) requires the accessory protein Vpx (). Vpx is essential for both SIV infection of primary macrophages and viral pathogenesis in vivo (). Vpx is incorporated into viral particles, suggesting that it might act immediately after viral fusion (). Viral reverse transcription is prevented in primary macrophages when cells are infected with either Vpx-deficient SIVor HIV-2 (). Interestingly, Vpx also increases the ability of HIV-1 to efficiently infect macrophages, dendritic cells, and resting CD4T cells when Vpx is incorporated into HIV-1 particles or supplied in trans (). Recent work identified SAMHD1 as the protein that blocks infection of SIVΔVpx, HIV-2ΔVpx, and HIV-1 before reverse transcription in macrophages, dendritic cells, and resting CD4T cells (). Mechanistic studies have suggested that Vpx induces the proteasomal degradation of SAMHD1 (). In agreement with this, the C-terminal region of SAMHD1 contains a Vpx binding motif, which is important for the ability of Vpx to degrade SAMHD1 (). SAMHD1 is a deoxyguanosine triphosphate (dGTP)-regulated deoxynucleotide triphosphohydrolase (dNTPase) that decreases the overall cellular levels of dNTPs (). Taken together, these results suggested that an overall decrease on the level of dNTPs is responsible for the block imposed on lentiviral reverse transcription.

Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses.

Covalent capture of kinase-specific phosphopeptides from total HeLa extracts revealed that a peptide derived from SAMHD1 containing T592 is the substrate for the human cdk1 complex (). These experiments suggested that human cdk1 might be the cellular kinase of SAMHD1. In agreement, analysis of the SAMHD1 protein sequence with the software Scansite ( http://scansite.mit.edu ) revealed that residuesTPQKconstitute a consensus sequence motif for recognition and phosphorylation by cdk1 (). To test the role of cdk1 in phosphorylation of SAMHD1, we assayed the level of SAMHD1 phosphorylation in the presence of the human dominant-negative cdk1 mutant cdk1-D146N, which is a cdk1-defective variant on its active site (). As shown in Figure 6 A, the presence of Cdk1-D146N decreased the amount of phosphorylated SAMHD1, suggesting that human cdk1 is phosphorylating SAMHD1 in vivo. To directly test the ability of human cdk1 to phosphorylate SAMHD1, we incubated bacterially expressed recombinant glutathione S-transferase (GST)-SAMHD1 with an active cdk1 complex in an in vitro kinase assay ( Figure 6 B). These experiments showed that cdk1 phosphorylates GST-SAMHD1, but not the GST control protein. We performed a similar in vitro kinase assay using the human histone H1, which is a specififc substrate for the cdk1 complex, as a positive control. These results, together with the evidence that SAMHD1 variants on T592 are not phosphorylated in vivo ( Figures 3 A and 3B), suggest that SAMHD1 is a substrate for the kinase complex cdk1. The results are also in agreement with the observation that mutating P593A in the cdk1 recognition motifTPQKabrogates SAMHD1 phosphorylation ( Figure 3 ). The SAMHD1-P593A protein behaved similarly to SAMHD1-T592A/V, suggesting that this mutant lost its ability to be regulated by phosphorylation ( Figures 3 and 5 ).

(B) In vitro phosphorylation of recombinant GST-SAMHD1 purified from bacteria by cdk1 complex, which was purified from baculovirus-infected cells using γ-[ 32 P]ATP as a phosphate donor. As a positive control, we incubated the cdk1 kinase complex with the human histone H1, which is a known substrate for this kinase complex. As a negative control, we incubated a similar amount of purified GST protein with the kinase complex cdk1.

(A) Effect of the Cdk1 dominant-negative mutant D146N (Cdk1-D146N) on the phosphorylation levels of SAMHD1. Cell lysates from human 293T cells transfected with plasmids expressing the SAMHD1-FLAG and Cdk1-D146N-HA proteins were separated by SDS-PAGE containing Phos-tag (+Phos-tag). SAMHD1-FLAG and Cdk1-D146N-HA were detected by western blotting using anti-FLAG and anti-HA antibodies, respectively. As a control, the levels of SAMHD1 phosphorylation were determined in the presence of the empty vector pCMV. Protein samples were also separated in SDS-PAGE gels without Phos-tag (−Phos-tag). Loading was normalized by western blotting using GAPDH antibodies. Similar results were obtained in three independent experiments, and a representative experiment is shown.

To directly analyze the enzymatic activity of SAMHD1 phosphorylation variants, we tested the ability of immunoprecipitated SAMHD1 variants ( Figure 5 B) to hydrolyze α-P-labeled thymidine triphosphate (α-[P]TTP) to deoxythymidine (dT) and α-[P]PP, in the presence of the allosteric activator dGTP ( Figure 5 C). For this purpose, we incubated the indicated SAMHD1 variant in the presence of radio-labeled α-[P]TTP. Reaction products were separated using thin-layer chromatography in order to determine the amount of hydrolyzed α-[P]PP ( Figure 5 C), as previously shown (). In agreement, immunoprecipitated SAMHD1-T592D and SAMHD1-T592E exhibited similar enzymatic activity when compared to wild-type SAMHD1 ( Figure 5 C). As a control, we measured the enzymatic activity of the different SAMHD1 variants in the presence of an analog for dsRNA, which inhibits the enzymatic activity of SAMHD1 (). These experiments showed that the enzymatic activity of SAMHD1 phosphorylation variants is not affected. We also confirmed these results by measuring nucleotide hydrolysis via high-pressure liquid chromatography (HPLC) ( Figure S4 A), as described in Experimental Procedures . Overall, these findings suggest that the enzymatic activity of SAMHD1 is not sufficient for restriction of HIV-1. Similarly, we measured the ability of SAMHD1-T592D purified from insect cells to hydrolyze α-[P]TTP in the presence of the allosteric activator dGTP ( Figures 5 D, 5E S4 B, and S4C), as previously shown (). In agreement, purified SAMHD1-T592D showed similar α-[P]TTP hydrolyse activity when compared to the wild-type purified protein ( Figures 5 D, 5E, and S4 C). In the same way, we confirmed these results by measuring nucleotide hydrolysis via HPLC ( Figure S4 B).

Previous observations have suggested that SAMHD1 blocks HIV-1 replication by decreasing the intracellular pool of dNTPs (). A decrease in cellular dNTPs will prevent the occurrence of retroviral reverse transcription, a process that requires dNTPs. Given that SAMHD1 phosphorylation variants did not show an obvious defect of the known properties of SAMHD1, we decided to test whether the loss of restriction by SAMHD1 phosphorylation variants correlated with a gain in cellular dNTP levels. For this purpose, we measured the intracellular levels of dNTPs in differentiated U937 cells stably expressing the different SAMHD1 phosphorylarion variants ( Figure 5 A), as described previously (). Remarkably, U937 cells stably expressing SAMHD1 variants wherein T592 was replaced by a phosphomimetic residue (D or E) showed dNTP levels comparable to those of U937 cells stably expressing wild-type SAMHD1. In agreement with this, the cellular dNTP levels of U937 cells stably expressing the SAMHD1 construct 112-626-T592D, which loses the ability to block HIV-1 infection, were similar to the levels found in U937 cells stably expressing wild-type SAMHD1 ( Figure S3 and Table 1 ). These results indicate that the inability of SAMHD1-T592D, SAMHD1-T592E, and 112-626-T592D to block HIV-1 infection is not due to a defect in the dNTPase activity of the protein. These findings indirectly suggest that the dNTPase activity of SAMHD1-T592D, SAMHD1-T592E, and 112-626-T592D is intact.

(E) Similarly, the dTTPase activity of baculovirus recombinant SAMHD1 and SAMHD1-T592D proteins was determined by measuring the hydrolysis of radio-labeled α-[P]dTTP of the indicated recombinant SAMHD1 protein. Reactions were stopped at the indicated times and separated by thin-layer chromatography using polyethyleneimine cellulose, as described in Experimental Procedures . See also Figure S4 . A representative result of three independent experiments is shown.

(C) Thin-layer chromatography analysis of the dTTP-triphosphohydrolase activity of the different immunoprecipitated SAMHD1 variants. For this purpose, we incubated radio-labeled α-[ 32 P]dTTP in the presence of the indicated SAMHD1 variants. Products from the α-[ 32 P]dTTP hydrolysis were separated by thin-layer chromatography using polyethyleneimine cellulose. Hydrolysis of α-[ 32 P]dTTP yields dT and α-[ 32 P]PP, which are visualized by using a phosphoimager. As a control, we also measured the dTTP-triphosphohydrolase activity in the presence of the dsRNA analog ISD-PS. The results of three independent enzymatic reactions per treatment are shown.

(A) Quantification of dATP, dTTP, and dGTP levels from PMA-treated U937 cells expressing the indicated SAMHD1 variants was performed with a primer-extension assay, as described in Experimental Procedures . Similar results were obtained in three separate experiments, and a representative experiment is shown.

Because our observations suggested that the phosphorylation state of T592 regulates the ability of SAMHD1 to block retroviral restriction, we tested the role of the N-terminal residues (1–112) of SAMHD1 ( Figure 1 B) in the ability of T592 phosphorylation to regulate restriction. Our previous observations suggested that a construct containing SAMHD1 residues 112–626 is sufficient for potent restriction of HIV-1 (). To test the role of residues 1–112 in SAMHD1 regulation by phosphorylation, we created a construct containing the residues 112–626 of SAMHD1 wherein T592 was replaced by the phosphomimetic residue D (112-626-T592D). The phosphorylation levels of 112–626 and 112-626-T592D were measured in human 293T cells via western blotting using SDS-PAGE gels containing Phos-tag ( Figure 4 A). In agreement with our findings in the full-length SAMHD1, the protein composed of residues 112–626 was phosphorylated ( Figure 4 A). Interestingly, 112-626-T592D revealed an unphosphorylated protein similar to the full-length SAMHD1-T592D in noncycling cells ( Figure 4 A). To test the ability of 112-626-T592D to block HIV-1 infection, we stably expressed it in U937 cells ( Figure 4 B) and challenged cells with increasing amounts of HIV-1-GFP ( Figure 4 C). Interestingly, 112-626-T592D completely lost its ability to block HIV-1 infection when compared to 112–626 or full-length SAMHD1 ( Figure 4 C). Our results indicate that the SAMHD1 N-terminal residues 1–112 are dispensable for the ability of the T592 phosphorylation to regulate retroviral restriction.

(C) Human monocytic U937 cells stably expressing the different SAMHD1 variants were challenged with increasing amounts of HIV-1-GFP. Infection is shown as the percentage of GFP-positive cells 48 hr postinfection as measured by flow cytometry. As a control, U937 cells stably transduced with the empty vector pLPCX were challenged with increasing amounts of HIV-1-GFP. See also Figure S3 . Experiments were performed in triplicate, and a representative result is shown.

(B) Human monocytic U937 cells were stably transduced with wild-type, and the indicated SAMHD1 variants were analyzed for SAMHD1 expression by western blotting using anti-FLAG antibodies. Western blot analysis of GAPDH was used as a loading control.

(A) Human 293T cells were transfected with the indicated SAMHD1 variant. Cells were lysed 24 hr after transfection, and protein samples were separated using SDS-PAGE gels containing Phos-tag (+Phos-tag). SAMHD1 variants were detected by western blotting using anti-FLAG antibodies. Protein samples were also separated in SDS-PAGE gels without Phos-tag (−Phos-tag). As a loading control, cell lysates were western blotted using GAPDH antibodies.

Finally, we tested the cellular localization of the different SAMHD1 phosphorylation variants in human HeLa cells ( Figure S2 I), as described previously (). These results showed that all the studied SAMHD1 phosphorylation variants localized to the nuclear compartment, similarly to the wild-type SAMHD1 protein ( Figure S2 I). Overall, these results demonstrated that the SAMHD1 phosphorylation variants used in these studies do not exhibit a major defect in the different properties of SAMHD1.

We and others have previously demonstrated the ability of SAMHD1 to bind RNA (); therefore, we tested the ability of the different SAMHD1 phosphorylation variants produced in human 293T cells to bind RNA ( Figure S2 H), as described previously (). As shown in Figure S2 H, all the studied SAMHD1 phosphorylation variants showed the ability to bind the double-stranded RNA (dsRNA) analog phosphorothioate-containing interferon-stimulatory DNA (ISD-PS) as strongly as the wild-type SAMHD1 protein.

Allosteric regulation of the enzymatic activity of SAMHD1 occurs in the dimerization interface of the HD domain (). In agreement with this, we found that the SAMHD1 variant HD206AA is partially affected in its ability to oligomerize, which suggests that the defect in the ability of HD206AA to restrict HIV-1 might also be due to an oligomerization deficiency (). To evaluate the possibility that the phosphorylation variants of SAMHD1 lose restriction due to an oligomerization defect, we tested the ability of the different SAMHD1-FLAG variants to oligomerize with the SAMHD1-hemagglutinin (HA) corresponding variant ( Figure S2 G). Interestingly, we found that SAMHD1 variants that lost the ability to block retroviral infection did not lose oligomerization ability, suggesting that the inability of SAMHD1 variants to restrict retroviral infection is not due to a defect in oligomerization.

Finally, we tested the ability of Vpx from the ROD strain of HIV-2 (Vpx) to degrade the different SAMHD1 phosphorylation variants. As shown in Figure S2 F, Vpxdegraded the different SAMHD1 phosphorylation variants equally, suggesting that the ability of Vpxto degrade SAMHD1 is independent of the phosphorylation state of SAMHD1.

Because our results indicated that the unphosphorylated form of SAMHD1 blocks HIV-1 infection in noncycling cells, we tested the ability of SAMHD1-T592V, which could not be phosphorylated, to restrict HIV-1 in cycling cells. As shown in Figures S2 C and S2D, expression of SAMHD1-T592V in HeLa cells did not block HIV-1 infection. However, when SAMHD1-T592V was tested in cycling U937 cells, it showed a mild effect on HIV-1 infection ( Figure S2 E).

Our initial mass-spectrometry analysis of endogenously expressed SAMHD1 in THP-1 cells revealed that a small fraction of SAMHD1 was phosphorylated in position S33, and this pattern did not change when comparing cycling with noncycling cells ( Figure 1 A). To understand the contribution of S33 phosphorylation in the ability of SAMHD1 to restrict HIV-1 infection, we replaced S33 with a phosphomimetic (D) or an unphosphorytable (A) residue. SAMHD1 variants were stably expressed in U937 cells ( Figure S2 A), and the ability of these variants to restrict HIV-1-GFP was measured ( Figure S2 B). As shown in Figure 3 E, changes on S33 did not affect the ability of SAMHD1 to block HIV-1 infection.

To test the ability of SAMHD1 variants to restrict HIV-1 infection in noncycling cells, we stably expressed SAMHD1 variants in U937 cells ( Figure 3 C) and tested the ability of these cell lines to restrict increasing amounts of HIV-1 expressing GFP as a reporter for infection (HIV-1-GFP), as previously described (). As shown in Figure 3 D and Table 1 , SAMHD1 containing a phosphomimetic residue such as D or E lost the ability to block retroviral restriction in noncycling U937 cells, suggesting that SAMHD1 phosphorylated at position T592 is unable to block retroviral infection. By contrast, SAMHD1 variants wherein T592 was replaced by an unphosphorytable residue such as V or A were not affected in their ability to block infection by HIV-1-GFP ( Figure 3 D and Table 1 ). Similar restriction patterns were observed when using HIV-2, SIVΔVpx, feline immunodeficiency virus, equine infectious anemia virus, bovine immunodeficiency virus, and N-tropic and B-tropic murine leukemia viruses (data not shown), as described in. These results suggest that a phosphorylated SAMHD1 in position T592 is unable to restrict infection by different retroviruses.

Our biochemical analysis suggested that phosphorylated SAMHD1 in cycling cells does not restrict retroviral infection. To test the regulation of SAMHD1 restriction by phosphorylation, we generated a series of SAMHD1 phosphorylation variants wherein T592 was replaced by a phosmimetic (D and E) or unphosphorytable (V and A) residue ( Table 1 ). We initially analyzed the phosphorylation state of the different variants via western blotting using SDS-PAGE gels containing Phos-tag in human 293T cells ( Figure 3 A). Interestingly, SAMHD1 variants, wherein T592 was replaced by a phosphomimetic or an unphosphorytable residue, are not phosphorylated when compared to the wild-type SAMHD1, suggesting that T592 is the residue phosphorylated in human cells ( Figure 3 A). To confirm that these mutants were no longer phosphorylated in 293T cells, we tested whether these proteins are recognized by the anti-phospho-T592-SAMHD1 antibody. As shown in Figure 3 B, the wild-type SAMHD1 is recognized by the anti-phospho-T592-SAMHD1 antibody. By contrast, all variants wherein T592 was replaced by a different residue were not recognized by anti-phospho-T592-SAMHD1.

(D and E) PMA-treated human monocytic U937 cells stably expressing the indicated SAMHD1 variants were challenged with increasing amounts of HIV-1-GFP. Infection is shown as the percentage of GFP-positive cells 48 hr postinfection measured by flow cytometry. As a control, U937 cells stably transduced with the empty vector pLPCX were challenged with increasing amounts of HIV-1-GFP. See also Figure S2 . Experiments were performed in triplicate, and a representative result is shown. WT, Wild-type.

(C) Human monocytic U937 cells were stably transduced with wild-type SAMHD1 and the indicated SAMHD1 variant. PMA-treated stable cells were analyzed for SAMHD1 expression by western blotting using anti-FLAG antibodies. Similarly, western blot analysis of GAPDH was used as loading control.

(B) The different SAMHD1 variants expressed in 293T cells were also analyzed by western blotting using anti-phospho-T592-SAMHD1 antibodies, which only recognize a phosphorylated SAMHD1 protein at residues T592.

(A) Human 293T cells were transfected with wild-type or the indicated SAMHD1 variant. Cells were lysed 24 hr after transfection, and protein samples were separated using SDS-PAGE gels containing Phos-tag (+Phos-tag). SAMHD1 variants were detected by western blotting using anti-FLAG antibodies. The indicated sample was treated with λPP for detecting the migration of the unphosphorylated SAMHD1. In parallel, we performed a similar analysis using the empty vector pLPCX. Protein samples were also separated in SDS-PAGE gels without Phos-tag (−Phos-tag). As a loading control, cell lysates were western blotted using GAPDH antibodies.

The enzymatic activity of the different SAMHD1 variants was measured by a dTTP hydrolysis reaction, as described in Experimental Procedures . “+” indicates wild-type levels of activity.

The cellular dATP levels of PMA-treated U937 cells stably expressing the different SAMHD1 variants were determined by primer extension as described in Experimental Procedures . “Low” indicates similar to the dATP levels observed in PMA-treated U937 cells stably expressing wild-type SAMHD1.

Subcellular localization of the different SAMHD1 variants in HeLa cells was performed as described in Experimental Procedures . “N” indicates nuclear localization; “C” indicates cytoplasmic localization.

SAMHD1-FLAG variants were assayed for their ability to bind the dsRNA analog ISD-PS, as described in Experimental Procedures . “+” indicates the RNA binding achieved by wild-type SAMHD1.

Oligomerization of the different SAMHD1 variants was determined by measuring the ability of the SAMHD1-FLAG variant to interact with the corresponding SAMHD1-HA variant, as described in Experimental Procedures . “+ ” indicates 100% oligomerization, which corresponds to the amount of wild-type SAMHD1-HA that interacts with wild-type SAMHD1-FLAG.

HIV-1 restriction was measured by infecting U937 cells stably expressing the indicated SAMHD1 variants with HIV-1-GFP. After 48 hr, the percentage of GFP-positive cells (infected cells) was determined by flow cytometry.

a HIV-1 restriction was measured by infecting U937 cells stably expressing the indicated SAMHD1 variants with HIV-1-GFP. After 48 hr, the percentage of GFP-positive cells (infected cells) was determined by flow cytometry.

Finally, we analyzed the phosphorylation state of SAMHD1 in cycling and noncycling human monocytic U937 cells stably expressing SAMHD1-FLAG ( Figure 2 B). Similarly, noncycling U937 cells stably express SAMHD1-FLAG, which blocks retroviral replication and is unphosphorylated. Overall, our analysis revealed that in all studied noncycling cells that restrict retroviral infection, SAMHD1 is unphosphorylated, whereas cycling cells contain a phosphorylated SAMHD1.

We also analyzed the phosphorylation state of SAMHD1 in human primary MDMs. As suggested by our mass-spectrometry results, endogenously expressed SAMHD1 in human primary MDMs is unphosphorylated ( Figures 2 A and 2B).

Recent evidence demonstrated that SAMHD1 is responsible for the block imposed by resting CD4T cells to HIV-1 infection (); therefore, we tested the phosphorylation state of SAMHD1 in primary human resting and replicating CD4T cells ( Figure 2 A). In agreement with the hypothesis that unphosphorylated SAMHD1 is responsible for retroviral restriction, we found that resting CD4T cells exhibit an unphosphorylated SAMHD1 protein, whereas replicating CD4T cells exhibit a phosphorylated SAMHD1 protein ( Figure 2 A). To further test the phosphorylation state of SAMHD1 in resting CD4T cells, we developed a rabbit polyclonal antibody for a peptide derived from SAMHD1 containing a phosphorylated T592 (anti-phospho-T592-SAMHD1). In agreement with the Phos-tag results, we found that anti-phospho-T592-SAMHD1 only recognized the SAMHD1 protein from replicating CD4T cells ( Figure 2 C). These results are in agreement with our hypothesis that the unphosphorylated form of SAMHD1 is responsible for restriction.

Because endogenously expressed SAMHD1 in THP-1 cells only restricts when THP-1 cells are differentiated to a noncycling state by using PMA, we compared the phosphorylation state of SAMHD1 between cycling and noncycling THP-1 cells ( Figure 2 A). In agreement with our mass-spectrometry analysis, we found that noncycling cells contained an endogenously expressed, unphosphorylated SAMHD1 protein. On the contrary, SAMHD1 proteins from cycling THP-1 cells were phosphorylated. This evidence suggests that unphosphorylated SAMHD1 in noncycling THP-1 cells blocks retroviral infection.

Next, we analyzed the phosphorylation of SAMHD1 in HeLa cells ( Figure 2 A). Similarly, SAMHD1-FLAG transfected or stably expressed in HeLa cells is phosphorylated, which is in agreement with the fact that HeLa cells by themselves or stably expressing SAMHD1 do not restrict retroviral infection (data not shown).

To analyze the phosphorylation state of SAMHD1 in different human cell lines and primary cells, we studied the phosphorylation of SAMHD1 by western blotting using SDS-PAGE gels containing Phos-tag, a ligand that shifts the mobility of phosphorylated proteins (). Initially, we analyzed transfected SAMHD1-FLAG in human 293T cells. As shown in Figure 2 A, SAMHD1-FLAG transfected in 293T cells showed a broad migration pattern, reflecting the different phosphorylated forms of SAMHD1-FLAG. By contrast, SAMHD1 treated with λprotein phosphatase (λPP) showed a faster-migrating, more compacted band revealing the unphosphorylated form of SAMHD1. This experiment showed that SAMHD1 is phosphorylated in human 293T cells, which is in agreement with the fact that 293T cells stably expressing SAMHD1-FLAG do not restrict retroviral infection.

(C) The phosphorylation state of SAMHD1 in resting and replicating CD4 + T cells from two donors was analyzed by using a specific antibody that recognizes a phosphorylated SAMHD1 at position T592 (anti-phospho-T592-SAMHD1). As a control, we analyzed the samples by western blotting using anti-SAMHD1 and anti-GAPDH antibodies. Similar results were obtained in three independent experiments, and a representative experiment is shown.

(B) Similarly, we analyzed the phosphorylation state of SAMHD1-FLAG stably expressed in U937 cells in the presence or absence of PMA. As a loading control, cell lysates were western blotted using GAPDH antibodies.

(A) 293T or HeLa cells transfected with a plasmid expressing SAMHD1-FLAG or pLPCX were lysed 24 hr after transfection and treated or not with λPP, which is a protein phosphatase with activity toward phosphorylated serine, threonine, and tyrosine residues. Protein samples were separated by SDS-PAGE containing Phos-tag (+Phos-tag), a ligand that shifts the mobility of phosphorylated proteins, and analyzed by western blotting using FLAG antibodies. We also analyzed the phosphorylation state of endogenously expressed SAMHD1 proteins using anti-SAMHD1 antibodies in different cells: human THP-1 cells, human primary MDMs, human primary resting CD4 + T cells, and replicating CD4 + T cells. Protein samples were also separated in SDS-PAGE gels without Phos-tag (−Phos-tag).

Our results suggest that unphosphorylated SAMHD1 on T592 in noncycling cells is antivirally active and that phosphorylated SAMHD1 on T592 in cycling cells is antivirally inactive ( Figure 1 B).

SAMHD1 is expressed in cycling and noncycling cells; however, the ability of SAMHD1 to block infection by different retroviruses is only observed in noncycling cells (). To find the molecular basis for this phenotype, we explored the state of SAMHD1 phosphorylation in cycling and noncycling cells by using mass spectrometry. Immunoprecipitated SAMHD1 proteins from cycling and noncycling cells were separated by SDS-PAGE and analyzed by mass spectrometry ( Figure S1 available online). Interestingly, we observed that SAMHD1 is phosphorylated at residue T592 when endogenously expressed in cycling THP-1 cells ( Figure 1 A). Similarly, cycling human monocytic U937 cells, which do not exhibit detectable levels of endogenous SAMHD1, engineered to stably express SAMHD1-FLAG (U937-SAMHD1-FLAG) revealed a phosphorylated SAMHD1-FLAG protein at residue T592 ( Figure 1 A). By contrast, noncycling cells, such as THP-1 and U937-SAMHD1-FLAG cells differentiated to a noncycling state by PMA, exhibited a SAMHD1 protein unphosphorylated at position T592 ( Figure 1 A). These results correlated the phosphorylation state of SAMHD1 with its antiviral activity ( Figure 1 A). Because these experiments suggested that unphosphorylated SAMHD1 is antivirally active, we tested the state of SAMHD1 phosphorylation in human primary monocyte-derived macrophages (MDMs). Similarly, we analyzed immunoprecipitated SAMHD1 from human primary MDM via mass spectrometry ( Figure S1 ). In agreement with our findings, human primary MDMs contained an unphosphorylated SAMHD1 in position T592 ( Figure 1 A). We also found that a small fraction of SAMHD1 was phosphorylated in position S33; however, this phosphorylation did not change when comparing cycling with noncycling cells ( Figure 1 ).

(B) Wild-type (WT) human SAMHD1 protein is depicted showing the numbers of the amino acid residues at the boundaries of each domain. The residue T592 is depicted in the consensus motif 592 TPQK 595 for recognition and phosphorylation by cdk1.

(A) Endogenously expressed SAMHD1 was immunoprecipitated using anti-SAMHD1 antibodies from PMA-treated (noncycling) or untreated (cycling) human monocytic THP-1 cells. Proteins were separated by SDS-PAGE, and a Coomassie-blue-stained band containing the SAMHD1 protein was subjected to phosphopeptide mapping by mass spectrometry. The ratios of phosphorylated to unphosphorylated peptides are shown. The ability of the different cells to restrict HIV-1 is presented. Similar analysis was performed for determining the phosphorylation state of the endogenously expressed SAMHD1 in human MDMs. In parallel, PMA-treated or untreated human monocytic U937 cells stably expressing SAMHD1-FLAG were used to immunoprecipitate and determine the phosphorylation state of SAMHD1, as described in Experimental Procedures . Mass-spectrometry analysis was performed three times, and a representative experiment is shown. See also Figure S1

Discussion

Cycling and noncycling cells have the potentiality to express the restriction factor SAMHD1; however, SAMHD1 is only antivirally active in noncycling cells, suggesting that differences exist between cycling and noncycling cells that might be regulating the ability of SAMHD1 to block retroviral infection. The first possible explanation is that SAMHD1 is posttranslationally modified when comparing cycling with noncycling cells. Second, noncycling cells might be expressing a cofactor required for restriction, and a third possibility would be that SAMHD1 posttranslational modifications and a cofactor are required for retroviral restriction. The present work revealed that the ability of SAMHD1 to restrict retroviral infection is regulated by phosphorylation of T592. Interestingly, we found that all tested cycling cells expressed a SAMHD1 protein phosphorylated at residue T592. By contrast, all tested noncycling cells revealed an unphosphorylated SAMHD1 protein at position T592.

The replacement of T592 with phosphomimetic amino acids completely abrogated the ability of SAMHD1 to block retroviral infection. SAMHD1 phosphorylation variants T592D and T592E completely lost their capacity to block retroviral infection without losing RNA binding, dNTPase activity, oligomerization, and localization. The fact that these variants did not lose most of the known SAMHD1 properties suggests that these variants are properly folded. One possibility is that phosphorylation of SAMHD1 at residue T592 induces a conformational change that eliminates the ability of SAMHD1 to block retroviral infection without affecting all the known properties of SAMHD1. A second possibility is that phosphorylation of residue T592 regulates the ability of SAMHD1 to interact with an unknown cofactor required for retroviral restriction.

White et al., 2013 White T.E.

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Diaz-Griffero F. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1. The ability of phosphorylation to regulate the restriction capacity of SAMHD1 only requires the HD domain and the C-terminal residues 583–626 ( Figure 1 B). Previous studies showed that a SAMHD1 construct containing only the HD domain and the C-terminal residues 583–626 (112–626) is sufficient for potent restriction of retroviruses (); this work suggested that the SAM domain is dispensable for restriction. Our investigations revealed that the SAMHD1 construct 112–626 wherein T592 has been replaced by a phosphomimetic residue loses its restriction capacity, suggesting that regulation of restriction by phosphorylation does not require the N-terminal residues 1–112. In agreement with this, 112-626-T592D lost the ability to block retroviral infection without losing dNTPase activity.

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Kim B.

Brojatsch J.

Diaz-Griffero F. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1. Recent observations have suggested that SAMHD1 exhibits 3′ to 5′ exonuclease activity against single-stranded DNA and RNA in vitro (). An alternative possibility is that phosphorylated SAMHD1 at position T592 exhibits low exonuclease activity without losing the ability to decrease the cellular levels of dNTPs. By contrast, a dephosphorylated SAMHD1 would exhibit high exonuclease activity. This would suggest that SAMHD1 is stopping viral replication by degrading the genetic material of the virus, which is in agreement with the ability of SAMHD1 to restrict diverse RNA viruses such as lentiviruses and γ-retroviruses (). Future experiments will attempt to understand whether the enzymatic activity of SAMHD1 is required for restriction.