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Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription.
Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment.
Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.
The human immunodeficiency virus type-1 (HIV-1) primarily infects CD4+ T-cells. While activated lymphocytes support viral replication, non-cycling quiescent CD4+ T-cells allow entry of HIV-1 but fail to allow efficient and complete reverse transcription [1-6]. However, addition of deoxynucleosides to unstimulated lymphocytes cultures partially overcomes this failure [7-9], suggesting that an insufficient supply of deoxynucleotide triphosphates (dNTPs) in quiescent T-cells may, at least in part, contribute to inefficient viral DNA synthesis [8,10]. Interestingly, the Aicardi-Goutières syndrome gene product SAMHD1 was recently described as the restriction factor that blocks HIV-1 infection of non-cycling myeloid cells [11-13]. SAMHD1 is a dGTP-dependent deoxynucleotide triphosphohydrolase [14-16] that reduces the cellular pool of dNTPs in differentiated, non-cycling myeloid cells to levels below those required to support HIV-1 DNA synthesis [15,17]. However, SAMHD1’s spectrum of activity beyond cells of the myeloid lineage remains unclear.
We first determined whether SAMHD1 was expressed in peripheral blood lymphocytes from healthy donors. Western blot analysis revealed that both CD4+ and CD8+ T-cells express SAMHD1 (Figure (Figure1a).1a). The expression levels of SAMHD1 in unstimulated CD4+ T-cells were similar to those found in myeloid cells, including CD14+ monocytes, monocytes-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) (Figure (Figure1a).1a). Moreover, flow cytometry analysis revealed that all circulating CD4+ T-cells subsets, including naïve (Tn; CD45RA+, CCR7+), central memory (Tcm; CD45RA-, CCR7+) and effector memory cells (Tem; CD45RA-, CCR7-), expressed high levels of SAMHD1 (Figure (Figure1b),1b), raising the possibility that SAMHD1 could affect the susceptibility of resting CD4+ lymphocytes to HIV-1 infection.
To test this hypothesis, we exposed unstimulated peripheral blood mononuclear cells (PBMCs) to viral-like particles containing the SIV accessory protein Vpxmac251 (VLP-Vpx), which counteracts SAMHD1-mediated restriction by triggering its proteasomal degradation [12,13]. PBMCs treated with VLP-Vpx or empty VLPs (VLP-Mock) were analyzed for SAMHD1 expression by flow cytometry. An average of 16% of VLP-Vpx treated resting CD4+ T-cells were SAMHD1 negative at 96hours post-exposure (Figure (Figure1c),1c), whereas most monocytes (90.2%) lost the expression of SAMHD1 after 48hrs of VLP-Vpx treatment (Additional file 1: Figure S1). The difference in Vpx-mediated loss of SAMHD1 in quiescent CD4+ T-cells and monocytes might result from discrepancies in nuclear/cytoplasmic exchange . Indeed, we have recently shown that Vpx-mediated degradation of SAMHD1 requires its nuclear export [19,20]. In support of this hypothesis, loss of SAMHD1 expression was observed in more than 80% of T-cell receptor (TCR)-stimulated CD4+ lymphocytes expressing Vpx (Figure (Figure1d).1d). We next evaluated the effect of SAMHD1 loss on HIV-1-resistance phenotype of resting CD4+ T-cells. Restriction of HIV-1 replication in quiescent T-cells has been attributed to blocks at both the reverse transcription step and viral gene expression (e.g.: lack of transcription factors such as NF-kB and CyclinT required for viral transcription) [21,22]. Thus, to bypass the transcriptional block, we performed single-round infection experiments using an HIV-1 based lentiviral vector carrying an EGFP cassette under the transcriptional control of the CMV promoter (HIV-CMV-EGFP). Unstimulated PBMCs isolated from healthy donors were exposed for 12hrs to VLP-Vpx or VLP-Mock and subsequently infected with HIV-CMV-EGFP. As expected, no EGFP was detected when cells were treated with VLP-Mock (Figure (Figure2a),2a), while, VLP-Vpx treatment resulted in EGFP expression in quiescent (HLA-DR-, CD69-) CD4+ T-cells (Figure (Figure2a).2a). An average of 14% of VLP-Vpx treated resting CD4+ T lymphocytes expressed EGFP, while less than 1% of cells exposed to VLP-Mock were EGFP+ (Figure (Figure2a2a and Additional file 1: Figure S2a). As a control, VLP-Vpx treatment enhanced the permissiveness of CD14+ monocytes to HIV-CMV-EGFP (Additional file 1: Figure S3b). These results indicate that Vpx could alleviate the post-entry block to HIV-1 infection of unstimulated T-cells. Importantly, VLP-Vpx treatment was not associated with CD4+ T-cell activation (Additional file 1: Figure S2c) or proliferation (Figure (Figure2b).2b). Vpx increased the susceptibility of non-cycling quiescent T-cells (eFluorHigh) (Figure (Figure2b)2b) while it had no effect on the permissiveness of activated dividing T-cells (eFluorLow) despite high level expression of SAMHD1 (Additional file 1: Figure S2d). In addition, Vpx increased HIV-1 infection of all resting CD4+ T subsets, including highly refractory naïve cells (Tn) (Figure (Figure22c).
We then focused our attention on viral reverse transcription, which is initiated in most HIV-1 exposed T-cell subsets . Completion of this step is, nonetheless, reached in resting CD4+ T-cells at a much slower rate [1-6]. We asked whether SAMHD1 is responsible for the efficient reverse transcription block in resting T-cells, as it has been demonstrated for myeloid cells [13,17]. PBMCs were exposed to VLP-Vpx or VLP-Mock and infected with HIV-CMV-EGFP. The kinetics of reverse transcription leading to production of full-length HIV-1 DNA in unstimulated CD4+ T-cells was determined by quantitative PCR. We observed that both the amount and the rate of reverse transcription leading to the production of full length viral DNA were enhanced in Vpx treated resting CD4+ T-cells compared to VLP-Mock treated counterparts (Figure (Figure2d).2d). These results show that VLP-Vpx overcomes the restriction of HIV-CMV-EGFP infection in resting CD4+ T-cells by promoting the accumulation of full length reverse transcripts. We next verified whether the observed effect of Vpx applies to wild type HIV-1. For this purpose, unstimulated PBMCs were treated with VLP-Mock or with VLP-Vpx and subsequently infected with HIV-1 expressing EGFP (HIV-EGFP) (Figure (Figure2e).2e). Following infection, we did not detect significant EGFP expression in both VLP-Mock- and VLP-Vpx-treated resting CD4+ T-cells (Figure (Figure2e2e and Additional file 1: Figure S3a). This is consistent with the HIV-1 LTR transcriptional block associated with their quiescent status and confirms that the analyzed CD4+ T-cells are indeed in a resting state. As a control, VLP-Vpx enhanced the permissiveness to HIV-EGFP of CD14+ monocyte population (Figure (Figure2e2e and Additional file 1: Figure S3a). A potential infectivity defect was ruled out, since TCR-mediated activation of T-cells efficiently induced EGFP expression (Additional file 1: Figure S3b). Interestingly, while no EGFP positive cells were detected in resting CD4+ T-cells, an accumulation of HIV-1 full length DNA was observed in VLP-Vpx treated cells (Figure (Figure2e).2e). Thus, Vpx promotes the accumulation of full-length viral DNA following the infection of resting CD4+ T-cells, but does not relieve the transcriptional block required for viral gene expression. The ability of Vpx to promote infection was further confirmed in another model of resting lymphocytes (Additional file 1: Figure S4). Purified CD4+ T cells were activated with PHA and cultured in IL-2 for 14-20days, until disappearance of the CD69 and Ki67 activation markers . Treatment of such cells with VLP-Vpx induced SAMHD1 loss in a large fraction of the cells, and a 6-fold increase in their sensitivity to HIV-CMV-GFP infection (Additional file 1: Figure S4). Importantly, the majority of infected EGFP-positive cells were found in the SAMHD1-negative cell subset (Additional file 1: Figure S4). Taken together, these results indicate that Vpx, acting through SAMHD1, facilitates infection of resting CD4+ T-cells by promoting the accumulation of fully reverse transcribed viral DNA in quiescent lymphocytes.
To confirm the role of SAMHD1 in the ability of Vpx to overcome HIV-1 restriction in quiescent CD4+ T-cells, we used PBMCs isolated from 4 Aicardi-Goutières syndrome patients harboring homozygous inactivating mutations in the SAMHD1 gene (AGS-5, referred to as SAMHD1-/-) . Heterozygous donors for these SAMHD1 mutations (referred to as SAMHD1-/+) were used as controls. We first assessed the intrinsic susceptibility of unstimulated SAMHD1-/- and SAMHD1-/+ CD4+ T-cells to HIV-CMV-EGFP infection. While heterozygous deletion of SAMHD1 did not affect the intrinsic resistance of unstimulated PBMCs to HIV-CMV-EGFP infection, homozygous deletion increased the susceptibility of both quiescent CD4+ T-cells and monocytes (Figure (Figure3a,3a, b, c), indicating that SAMHD1 is required to mediate HIV-1 restriction in resting CD4+ T-cells. Remarkably, VLP-Vpx treatment did not further enhance permissiveness of SAMHD1-/- resting CD4+ T-cells (Figure (Figure3b,3b, d). However, the restrictive phenotype of SAMHD1-/+ cells is alleviated after VLP-Vpx delivery (Figure (Figure3a,3a, d), indicating that SAMHD1 is required for Vpx to overcome HIV-1 restriction in T-cells. Overall, these results demonstrate that SAMHD1 acts as an effective HIV-1 restriction factor in non-cycling resting CD4+ lymphocytes.
The demonstration that SAMHD1 restricts HIV-1 replication in quiescent CD4+ T-cells could have an important implication in our understanding of HIV-1-mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks. It has recently been shown that abortive HIV-1 reverse transcription in resting CD4+ T-cells leads to the accumulation of cytoplasmic viral nucleic acids that trigger a host defense program eliciting a coordinated proapoptotic and proinflammatory response [25,26]. By preventing completion of reverse transcription in quiescent CD4+ T-cells, SAMHD1 could contribute to their depletion. The HIV-1 reservoir, which mainly consists of quiescent CD4+ T-cells that harbor integrated silent provirus, represents a major barrier to viral eradication by antiretroviral therapy. It has recently been shown that chemokines can facilitate early steps of HIV-1 replication in resting CD4+ T-cells, leading to latency . It will be of importance to determine whether chemokines regulate SAMHD1 activity and facilitate the generation of latently infected cells in vivo. The regulation of SAMHD1 activity remains an important area of study. In this regard, we observed that SAMHD1 restriction activity does not correlate with its expression levels. Indeed, although SAMHD1 expression is independent of the activation state of CD4+ T-cell, its restriction activity is witnessed only when the cells are in a quiescent state (Additional file 1: Figure S2a and S2d). SAMHD1 activity could be regulated through post-translational modifications and/or through the expression of a cellular partner in non-cycling cells, including quiescent CD4+ T-cells. Additionally, SAMHD1 activity can also be regulated through the expression of splice variants lacking the enzymatic activity . Given that dN supply only partially rescues HIV-1 reverse transcription in resting CD4+ T-cells, one can ask whether the restriction imposed by SAMHD1 is fully or partially due to its dNTP triphosphohydrolase activity. Interestingly, it has recently been shown that SAMHD1 is a nucleic acid binding protein that displays a preference for RNA over DNA . Further studies are required to elucidate the mechanism by which SAMHD1 restricts HIV-1 in resting CD4+ T-cells. Deciphering the functional interaction between HIV-1 and SAMHD1 will lead to a better understanding of the damage imposed by this virus to the immune system and the progression towards AIDS.
CD14+ and CD4+ T-cells were purified using CD14+ microbeads or CD4+ T-cell isolation kit (Miltenyi Biotec). CD14+ monocytes were treated 2hours with VLP-Vpx before preparation of whole cell extracts (WCE). WCE were prepared with buffer containing 0.5% Triton X-100, 150mM NaCl, 10mM KCL, 1.5mM MgCl2, 0.5mM EDTA 10mM β-mercaptoethanol, 0.5mM PMSF. Cell lysates were boiled in SDS sample buffer and resolved on a SDS-PAGE gel (Biorad). Proteins were liquid-transferred (Biorad) to nitrocellulose membrane in transfer buffer (20% methanol, 25mM Tris, 192mM Glycine, 0.037% SDS) during 90min at 100V. Western blotting was performed using the following antibodies: mouse anti-SAMHD1 (Abcam #AB67821) and rabbit anti-Erk1/2 (Cell Signaling Technology).
Purified CD4+ T-cells were stimulated for 3days with anti-CD3, anti-CD28 and IL-2 and transduced with Vpx expressing retroviral construct . Two days post transduction, cells were harvested and fixed in PBS with 4% paraformaldehyde and 2% sucrose, and permeabilized with 0.5% Triton X-100, 20mM Tris (pH 7.6), 50mM Nacl, 3mM MgCl2, and 300mM sucrose. Wash and antibody incubation steps were performed in PBS-0.1%Tween. Cells were stained with Anti-SAMHD1 (Abcam #AB67821) and Vpx was stained with anti-HA (Covance). Secondary antibodies were purchased from Invitrogen. Nuclei were stained with DAPI in mounting media (Vectashield; Vector Labs) and images were collected on a Zeiss Axioimager Apotome.
SIV3+ was kindly provided by N. Manel. SIV- was a gift from J. Luban. HA-Vpxmac251 was subcloned in pOz-IL2Rα expression vector. pHRET was kindly provided by C. Mettling. pBR-NL4-3-IRES-EGFP was a gift of F. Kirchhoff. psPAX2 packaging plasmid was obtained from Addgene (D. Trono). MMLV packaging plasmid, A-MLV envelope and pMD2-G VSV-G envelope were previously described .
VLPs and viral particles were produced from 293T-cells using the standard phosphate calcium transfection protocol. For VLPs, 293T-cells were transfected with 8μg SIV3+and 2μg pMD2-G VSV-G encoding plasmid (VLP-Vpx) or with 8μg SIV- and 2μg pMD2-G (VLP-Mock). Media were replaced 16h after transfection, and VLPs were harvested 48h later, filtered at 0.45μm and concentrated 100 times by ultracentrifugation. For virus production, HIV-EGFP was produced by transfection of 10μg of pBR-NL4-3-IRES-EGFP and 1μg of pMD2-G. HIV-CMV-EGFP was produced by transfection of 5μg of pHRET, 5μg of psPAX2 packaging vector, and 2.5μg of pMD2-G. For MLV transduction particles, 293T-cells were transfected with 5μg pOz HA-Vpxmac251 construct, 2.5μg MMLV packaging plasmid, and 2.5μg A-MLV envelope encoding plasmid. When required, p24 concentration was measured by ELISA (Innogenetics).
PBMCs were isolated from blood samples by Ficoll gradient (Eurobio), cultured 12h in presence of VLP- Vpxmac251 or VLP-Mock at a density of 2 million cells per well (24 well plates) in 300μl of 10% FCS supplemented complete RPMI (R10) (Invitrogen). Infection was then performed by addition of 300μl of R10 diluted HIV-CMV-EGFP (1μg) or HIV-EGFP (800ng) for 4days before analysis. As control, TCR stimulated PBMCs were cultured for 3days on plate-bound anti-CD3 antibody (5μg/ml) (Miltenyi Biotec) in presence of 1μg/ml anti-CD28 antibody and IL-2 (20U/ml) (Roche) and infected with HIV-CMV-EGFP (1μg) or HIV-EGFP (800ng) for 2days before analysis. Optimal viral inoculums were determined by titration using TCR stimulated PBMCs. Alternatively, after thawing, Trypan Blue viability assessment and TURBOTM DNase (Ambion) treatment at 37°C for 1hour were performed, and cryopreserved PBMCs were cultured in aforementioned conditions.
Non-cycling, quiescent CD4+ T-cells and monocytes were analyzed using the following antibodies and dye: Brilliant-Violet421 anti-CD3 (UCHT1), PeCy7 anti-CD4 (RPA-T4), PE anti-CD69 (FN50), APC anti-HLA-DR (TU36), V500 anti-CD14 (M5ED), PeCy7 anti-CCR7 (3D12), PE anti-CD45RA (HI100) (BD Biosciences), PerCP anti-CD45 (5B1) (Miltenyi Biotec), Cell-Proliferation Dye eFluor® 670 (eBiosciences) and Alexa-488 Anti-SAMHD1 (I-1918) (O. Schwartz). Cells were analyzed on MACSQuant Analyzer (Milteny Biotec). Data analyses were performed on FlowJo (TreeStar inc).
Prior infection, viral stocks were treated for 1h at 37°C with 100U/ml of TURBOTM DNase (Ambion). PBMCs (2 X 106 cells) were infected with 1000ng of HIV-CMV-EGFP or with heat inactivated HIV-CMV-EGFP. CD4+ T-cells were purified from cultured PBMCs by depleting non CD4+ T-cell, using CD4+ T-cell isolation kit (Miltenyi Biotec) (Average purification yield of 97%). Genomic DNA was extracted using the QIAmp DNA blood minikit (Qiagen). Full-length viral DNA quantification was performed by quantitative PCR using primers annealing in the 5’LTR-U5 and gag regions. PCR measurements were performed in duplicate using SYBR Green (Qiagen). Amplifications were carried out in the LightCycler480 (Roche). The average of the technical duplicates was normalized to GAPDH levels using the comparative CT method (2ΔΔCT).
CD4+ T cells were isolated by positive selection as described above (Miltenyi Biotec). Resting CD4+ T cells were activated with 1ug/ml phytohemagglutinin (PHA) and 100 U/ml Interleukin 2 (IL-2) and cultured in fresh medium containing IL-2 for 14 to 20days . The activation state was monitored every few days by flow cytometry after staining with PE-coupled CD69 and FITC-coupled Ki67 (BD Pharmingen). Resting post-activated cells were treated with Vpx-VLPs or Mock-VLPs for 3hours, washed and incubated overnight with HIV-CMV-EGFP. The following day, cells were washed and incubated in fresh medium containing IL-2 for 96hours. Percentages of EGFP positive and SAMHD1 negative cells were then assessed by flow cytometry.
The authors declare that they have no competing interests.
BD performed most of the experiments. AC and CCB, performed some experiments. OS and DA designed and performed the experiment shown in Additional file 1: figure S4. GR and YC provided AGS5 cells. BD and MB designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
SAMHD1 restricts HIV-1 replication in quiescent CD4+ T-cells.
We thank Rosemary Kiernan and members of the Molecular Virology Lab for critical reading of the manuscript. We thank the Montpellier RIO imaging facility (MRI) for providing adequate environment for microscopy experiments. We thank ClÃ©ment Mettling for providing us pHRET for HIV-CMV-EGFP production. This work was supported by grants from the ERC (250333), Sidaction (fonds de dotation Pierre Bergé), ANRS and FRM “équipe labéllisée” to MB. BD was supported by ERC fellowships; AC by ANRS, NL by SIDACTION and YA by MESR, Ecole de l’INSERM-Liliane Bettencourt and FRM fellowships. Work in OS lab was supported by ANRS, Sidaction, Areva, the Labex IBEID program, Areva, and the Vaccine Research Institute. DA was supported by ANRS.