SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4(+) T-cells

Abstract

Background: Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription.

Results: 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.

Conclusion: 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.

Figure 1

CD4 T lymphocytes express SAMHD1.a. Western blot analysis of SAMHD1 expression in primary hematopoietic cells. b. Flow cytometry analysis of SAMHD1 expression levels in CD14⁺ monocytes, T-cells (CD4⁺ and CD8⁺ T lymphocytes) and in CD4⁺ T-cell subsets including Naïve (Tn; CD45RA⁺, CCR7⁺), Central Memory (Tcm; CD45RA^-, CCR7⁺), and Effector Memory (Tem; CD45RA^-, CCR7^-). c. Kinetics of SAMHD1 loss in quiescent (CD69^-, HLA-DR^-) CD4⁺ T-cells after VLP-Vpx treatment (representative experiment, n = 2). d. Vpx transduction of activated CD4⁺ T-cells induces loss of SAMHD1. TCR-stimulated CD4⁺ T-cells were transduced with Vpx-HA expressing retroviral construct. Forty-eight hours later, SAMHD1 and Vpx were immunostained using specific antibodies. Nuclei were stained in mounting media with DAPI. Pie charts represent the proportions of SAMHD1⁺ cells among 100 Vpx positive and negative counted cells.

Figure 2

Vpx treatment of quiescent CD4⁺T-cells results in increased susceptibility to HIV-1 infection.a. Impact of Vpx treatment on quiescent CD4⁺ T-cells susceptibility to HIV-CMV-EGFP infection. Upper right panel: expression vectors used to produce HIV-CMV-EGFP virions. Left panel: PBMCs were treated 12 h with VLP-Mock or VLP-Vpx then infected with HIV-CMV-EGFP or uninfected (control). Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) were assessed 4 days post infection. Lower right panel: Results are expressed as percentage of EGFP positive cells. Graphic shows mean and standard deviation for 6 healthy donors. b. PBMCs were cultured as in a. after staining with the eFluor670 proliferation dye. Cell division and EGFP expression were assessed in CD4⁺ T-cells (a representative experiment is shown). c. Impact of Vpx on quiescent CD4⁺ T-cell subsets susceptibility to HIV-CMV-EGFP infection. Left panel: gating strategy to define CD4⁺ T-cell subsets: Naïve (Tn; CD45RA⁺, CCR7⁺), Central Memory (Tcm; CD45RA^-, CCR7⁺) and Effector Memory (Tem; CD45RA^-, CCR7^-). Middle panel: Permissiveness of Tn, Tcm and Tem to HIV-CMV-EGFP performed as described in a (a representative experiment; n = 3). d. Kinetics of full length HIV-CMV-EGFP DNA accumulation quantified by quantitative PCR in purified non-stimulated CD4⁺ T-cells from 2 healthy donors #5 and #6. Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) assessed by flow cytometry 4 days post infection by HIV-CMV-EGFP are indicated on the right. e. Effect of Vpx on the permissiveness of quiescent CD4⁺ T-cells to infection by HIV-EGFP. Upper panel: expression vectors used to produce HIV-EGFP virions. Lower left panel: PBMCs were cultured as in a. except cells were infected with HIV-EGFP. Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) and CD14⁺ monocytes were assessed 4 days post infection for 3 donors. Lower right panel: Full length HIV-EGFP DNA accumulation was performed as in d. for 1 donor.

Figure 3

Quiescent CD4⁺T-cells from Aicardi-Goutières Syndrome patients, with inactivating mutation ofSAMHD1(AGS-5), display a Vpx-independent susceptibility to HIV-1 infection.a. Impact of Vpx treatment on susceptibility to HIV-CMV-EGFP infection of quiescent CD4⁺ T-cells (CD69- HLA-DR-) and CD14⁺ monocytes from individuals with heterozygous AGS-5 SAMHD1^-/+ mutation (n = 2). PBMCs were treated 12 h with VLP-Mock or VLP-Vpx then infected with HIV-CMV-EGFP or left uninfected (control). Proportions of EGFP positive quiescent CD4⁺ T-cells were assessed 4 days post infection. b. PBMCs from AGS-5 SAMHD1^-/- patients were treated and analyzed as in a. (n = 4). c. Impact of SAMHD1 homozygous mutation on permissivity to HIV-CMV-EGFP infection of CD4⁺ T-cell (left panel) and monocytes (right panel). Results are expressed as a fold increase of GFP positive cells among quiescent CD4⁺ T-cells, comparing SAMHD1^-/- patients to SAMHD1^-/+ individuals. d. Effect of Vpx treatment on the permissiveness of quiescent CD4⁺ T-cell (left panel) and monocytes (right panel) derived from SAMHD1^-/- patients (n = 3) and SAMHD1^-/+ (n = 1) to HIV-CMV-EGFP. Results are presented as fold increase comparing the percentage of GFP positive cells in VLP-Vpx to VLP-Mock treated samples.