To test this hypothesis, we exposed unstimulated peripheral blood mononuclear cells (PBMCs) to viral-like particles containing the SIV accessory protein Vpx_mac251 (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 [18]. 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 (eFluor^High) (Figure ​(Figure2b)2b) while it had no effect on the permissiveness of activated dividing T-cells (eFluor^Low) 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 [6]. 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 [23]. 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^-/-) [24]. 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.