SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutières syndrome are highly susceptible to HIV-1 infection

Abstract

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.

Figure 1. SAMHD1 is identified as Vpx binding protein leading to its degradation.

A) pNTAP-SIV Vpx was transfected in 293T cells. After lysis, isolation of SIV-Vpx and binding proteins was performed via Tandem Affinity Purification. The proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Protein bands in which the respective protein was identified are indicated. B) Schematic representation of the Vpx protein indicating the helical structure elements (black) and the introduced T17A mutation. C) 293T cells were transfected with pcDNA3.1 (mock) or plasmids encoding HA-Vpx or HA-Vpx T17A. After 48 hours, the cells were lysed and subjected to HA-immunoprecipitation. Isolated proteins were separated via SDS-PAGE and analyzed with antibodies specific for HA or SAMHD1. D) HeLa cells endogenously expressing SAMHD1 were transfected with pcDNA3.1 (mock) or HA-Vpx or HA-Vpx T17A encoding plasmids. 24 hours after transfection, the cells were fixed and stained for SAMHD1 and HA-Vpx with fluorescent antibodies. Nuclei were stained with DAPI before performing confocal microscopy. White arrows indicate nuclei of Vpx expressing cells, in which SAMHD1 is absent. White bar length corresponds to 10 µm. E) THP-1 cells were incubated with 2 MOIeq of empty VLPs (Ø), Vpx-VLPs or Vpx T17A-VLPs in the presence or absence of 50 µM MG132. After 6 hours the cells were analyzed by western blot analysis with the indicated antibodies.

Figure 2. SAMHD1 is a novel interferon-induced restriction factor for HIV-1 in monocytic cells.

A) THP-1 cells were stimulated with PMA for 24 hours before they were transduced with empty VLPs (Ø) or VLPs carrying Vpx (VLP VPX) or Vpx T17A mutant (VLP Vpx T17A) with a MOIeq of 2. Two hours after transduction the cells were infected with HIV-1-luc. Luciferase activity was determined 24 hours after infection. In parallel, uninfected THP-1 cells were lysed six hours after VLP transduction and subjected to western blot analysis with the indicated antibodies (lower panel). B) THP-1 cells were transduced with lentiviral particles encoding for a non-specific shRNA (shControl) or a shRNA against SAMHD1 (shSAMHD1 #1; #2) and selected with puromycin. PMA stimulated THP-1 shControl or THP-1 shSAMHD1 (#1; #2) cells were infected with HIV-1-luc. After 24 hours, the luciferase activity was determined. In parallel, uninfected cells were subjected to western blot analysis using the indicated antibodies (lower panel). C) One day post-isolation, primary human monocytes were incubated with 2 MOIeq of VLPs containing no Vpx, WT Vpx or Vpx T17A and HIV-1-EGFP (MOI 8). EGFP expression was determined by FACS five days post infection. The experiment was repeated twice with different donors and the results of a representative experiment are shown here. D) Primary human monocytes were transduced with 2 MOIeq empty VLPs, Vpx VLPs or Vpx T17A VLPs and cultured w/o MG132 (50 µM) for 6 hours. The cells were lysed and analysed by immunoblot for expression of SAMHD1 and GAPDH. Mock indicates untransduced cells. E) Primary human monocytes were stimulated with 2, 20 or 200 ng/ml IFNα (ProSpec) for 24 hours or with 20 ng/ml IFN (Sigma) for 48 hours. The cells were lysed and SAMHD1 was detected with the respective antibody in western blot analysis.

Figure 3. PBMC from AIcardi-Goutières syndrome patients homozygous for SAMHD1 mutation are highly susceptible for spreading HIV-1 replication.

A) Schematic representation and domain structure of SAMHD1 and SAMHD1 R164X, indicating the truncation of SAMHD1 after insertion of a STOP codon observed in two AGS patients. B) PBMC from healthy donors (Donor 3/4 +/+) and AGS patients homozygous for SAMHD1 R164X (Donor 1/2 -/-) were isolated by Ficoll gradient, fixed and stained with PE-labeled anti-CD14, PE-labeled anti-CCR5, FITC-labeled anti-CD3 and PE-labeled anti-CD4 for FACS analysis. The numbers indicate the relative amount of the respective subpopulation within PBMC. C) A fraction of isolated PBMC described in (B) was stimulated with 250 ng/ml PHA & 180 U/ml IL-2 for 18 hours (left graph) or were left non-stimulated (right graph) prior to infection in triplicate with 0.05 MOI of HIV-1 SF162. At indicated days post-infection, the supernatants were analyzed for reverse transcriptase (RT) activity. D) A fraction of isolated Donor 2 (SAMHD1 -/-) and Donor 4 (SAMHD1 +/+) PBMC analyzed in (B) were infected in triplicate with HIV R7/3 YU2 EGFP or mock infected. At indicated days post-infection, the culture supernatants were analyzed for p24 concentration by ELISA.

Figure 4. Monocytic cells lacking SAMHD1 are target cells for HIV-1.

A) PBMC of the SAMHD1-deficient Donor 2 and the healthy Donor 4 were infected in triplicate with HIV-1 R7/3 YU2 EGFP as described in Figure 3D. After 7 days in culture, staining was performed with CD14-PE (red) and Hoechst 33342 (DNA staining, blue). GFP expression (green) indicates HIV infected cells. Images were acquired with 20x and 40x lenses on an Olympus microscope with live imaging system. B) Magnified selected composite image from infected cells derived from Donor 2 (SAMHD1 -/-). C) Magnified selected composite images from infected cells derived from Donor 2 (SAMHD1 -/-) and Donor 4 (SAMHD1 +/+). Arrows identify CD14+ (red), GFP/HIV+ (green) and CD14+/GFP/HIV-1 + cells (orange). D) Percentage of CD14+ and CD14- cells within HIV-1/GFP+ cells from Donor 2 (SAMHD1 -/-) determined by cell counting of 16 independent microscopy images.

Figure 5. Cytokine secretion of PBMC lacking SAMHD1 indicates an early inflammatory response during HIV-1 replication.

The culture supernatants of HIV-1 R7/3 YU2 EGFP infected and non-infected PBMC of the SAMHD1-deficient Donor 2 and the healthy Donor 4 as described in Figure 3D were subjected to Multiplex ELISA and analyzed for time-dependent secretion of the indicated chemokines and cytokines. The results are presented as cytokine concentrations for SAMHD1-deficient Donor 2 in the absence (purple lines) or presence (green lines) of HIV-1 infection and as fold induction upon HIV-1 infection over the mock infected controls for SAMHD1-deficient Donor 2 (yellow lines) and healthy Donor 4 (blue lines).