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. 2011 May 25;474(7353):654-7.
doi: 10.1038/nature10117.

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx

Nadine Laguette  1 , Bijan Sobhian, Nicoletta Casartelli, Mathieu Ringeard, Christine Chable-Bessia, Emmanuel Ségéral, Ahmad Yatim, Stéphane Emiliani, Olivier Schwartz, Monsef Benkirane
Affiliations

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx

Nadine Laguette et al. Nature. .

Abstract

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

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Figures

Figure 1
Figure 1. SAMHD1 interacts with Vpx and is degraded by the proteasome
a, SIVmac251 Vpx was tandem-affinity-purified from Flag- and HA-tagged Vpxmac251 (F/H-Vpx)-expressing THP-1 cells (THP-1-Vpx) and peptide-eluted under native conditions. Eluates were separated on SDS–PAGE and silver stained. SAMHD1 and major, previously described, Vpxmac251 interactants identified using tandem mass spectrometry are indicated (MW, protein molecular weight marker in kDa). b, Whole-cell extract (WCE) and Flag-immunoprecipitated F/H-Vpx analysis by western blot against DDB1, CUL4A and SAMHD1. c, THP-1 cells were treated with 50 μM MG132 for 2 h before a 2-h incubation with Vpxmac251 containing virus-like particles (VLP-Vpx). After a further overnight incubation with MG132, whole-cell extracts were prepared and analysed by western blot with the indicated antibodies. R.S.I., relative signal intensity. d, Analysis of the expression profile of SAMHD1 in different cell types by western blot. e, THP-1 cells were transduced with a bicistronic retroviral vector allowing expression of Vpxmac251, VpxROD, VpxRCM-NG and VpxRCM-GAB and a selectable marker. After cell sorting and whole-cell extraction, the ability of Vpx variants to degrade SAMHD1 in THP-1 whole-cell extract was analysed by western blot using the indicated antibodies.
Figure 2
Figure 2. SAMHD1 restricts HIV-1 infection in THP-1 cells
THP-1 cells were engineered to stably express shRNA 3 (3) or shRNA 4 (4) specifically targeting SAMHD1 (THP-1-shSAMHD1) or scrambled shRNA (THP-1-scr). a, THP-1-shSAMHD1 and THP-1-scr cells were infected with 50 ng of HIV-LUC-G. Luciferase activity was measured 24 h after infection and normalized for protein concentration in analysed samples. Results are expressed as fold increase of luciferase activity in THP-1-shSAMHD1 over THP-1-scr cells. N.I., non-infected. b, THP-1-shSAMHD1 and THP-1-scr cells were treated with VLP-Vpx before infection with 50 ng of HIV-LUC-G. Luciferase activity was measured as in a. Results are expressed as fold increase of luciferase activity in VLP-Vpx-treated over untreated cells. c, Mutants of Flag- and HA-tagged SAMHD1 (SAMHD1-F/H) were generated that are either shSAMHD1-resistant (SAMHD1-R) or mutated in the HD domain (SAMHD1(HD/AA)). These mutants were introduced in an MLV expression vector. Asterisk indicates synonymous mutation. d, THP-1-shSAMHD1 cells were transduced with SAMHD1-R for 48 h or left untreated, differentiated and infected with 100 ng of HIV-LUC-G. Luciferase activity was measured and expressed as in a. e, U937 myeloid cells were transduced with SAMHD1-F/H or SAMHD1(HD/AA) for 24 h. After a further 16-h differentiation step, cells were infected with 10 ng of HIV-LUC-G. Luciferase activity was measured as in a. Results are expressed as fold increase luciferase activity in transduced over parental U937 cells. f, Total viral DNA was quantified by quantitative PCR in THP-1-shSAMHD1 and THP-1-scr cells 24 h after infection with HIV-LUC-G or heat-inactivated virus (56 °C). Results are expressed as per cent maximum signal intensity. All graphs show mean ± standard deviation from a representative experiment (n = 5).
Figure 3
Figure 3. SAMHD1 restricts HIV-1 infection in primary MDDCs
a, THP-1 and monocyte-derived dendritic cells (MDDCs) from healthy donors (HD) were treated with VLP-Vpx for 2 h and infected with 100 ng of HIV-LUC-G. Luciferase activity was measured at 48 h after infection and normalized for protein concentration. Results are expressed as fold increase luciferase activity in VLP-Vpx treated over untreated cells. Graphs show mean ± standard deviation from a representative experiment (n = 4). b, Cells from a were subjected to whole-cell extraction before analysis by western blot using indicated antibodies. c, MDDCs from HD1 were mock transfected or transfected with siRNA targeting SAMHD1 (siRNA 1 and 2) or dynamin 2 (DYN2) for 48 and 96 h before whole-cell extraction and analysis by western blot using indicated antibodies. d, Cells from HD2 were transduced at 48 h with GFP encoding lentiviral vector (LV-GFP, 50 ng) and analysed by flow cytometry after 4 days. e, HD2 and HD3 were transduced as in d, except that a concentration of 5 ng of LV-GFP was used. The percentage of GFP-positive cells from HD2 and HD3 was quantified and expressed as fold increase GFP-positive cells relative to scrambled siRNA-treated cells. f, Transfected MDDCs from HD2 and HD3 were infected with 10 ng of HIV-G. Results are expressed as fold increase of p24-positive cells relative to scrambled siRNA-treated cells. g, MDDCs from HD2 and HD4 were transfected as in c before infection with 10 ng of luciferase-expressing lentiviral vector (LV-LUC). Results are expressed as in Fig. 2a.
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