Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response

Abstract

Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3' exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Figure 1. Murine SAMHD1 Functions to Reduce Cellular dNTP Concentrations

An assay based on incorporation of a spe-cific single nucleotide into a radiolabeled template by RT was used to quantify the relative amounts of each nucleotide in SAMHD1^Δ/Δ (KO) or control cells (WT). The indicated cell types were flow-cytometrically sorted (except for E14.5 MEFs), followed by extraction of dNTPs, which were subsequently used as substrates for the individual in vitro RT reactions. In a second independent experiment, two dNTP extracts of pools from peritoneal macrophages and one pool from bone-marrow-derived macrophages from SAMHD1^Δ/Δ or control cells yielded similar results. Displayed are the means ± SD of two independent measurements, each performed in triplicate.

Figure 2. Murine SAMHD1 Restricts Retro-viral Replication

(A) BMDCs were generated from six mutant and six control mice and infected with EGFP-containing HIV-1-VSV-G (VSVG/NL43-CMVGFP). Cellular EGFP fluorescence, indicating that the virus had successfully reverse transcribed and integrated its genome, was detected by FACS. The graph represents a summary of two independent experiments with similar results. A third experiment with cells from three additional mutant and three additional control mice yielded similar results (Figure S2). Means ± SD are displayed. (B) BMDCs from three SAMHD1-deficient mice were transduced with a lentiviral vector carrying either an EYFP and a murine SAMHD1 expression cassette or only the EYFP cassette. After 48 hr, the transduced cells were challenged with the EGFP-expressing HIV-1 pseudotyped with VSV-G (see A). EYFP⁺ cells were analyzed by FACS for EGFP expression 48 hr after challenge. Means ± SD are displayed. (C) Five SAMHD1^Δ/Δ and five SAMHD1^WT/WT mice were injected intravenously with 1.8 × 10⁷ particles of an EGFP-expressing HIV-1 pseudotyped with VSV-G (VSVG/HR.CMVGFP) and two control mice (Ctrl, one SAMHD1^Δ/Δ and one SAMHD1^WT/WT) were injected with PBS. After 3 days, the mice were sacrificed and the absolute numbers of live EGFP⁺ cells per 10⁶ splenocytes were determined by flow cytometry. Means ± SD are displayed. Differential analysis of individual cell populations from this experiment for HIV reporter infection is shown in Figure S2. (D) Infection of five additional SAMHD1^Δ/Δ and five additional WT mice with 5 × 10⁶ particles of HIV reporter virus as in (C). Absolute numbers of live EGFP⁺ cells in different splenic cell populations are shown. Means ± SD are displayed. See also Figure S2.

Figure 3. Spontaneous Induction of Type I IFN-Inducible Genes in SAMHD1-Deficient Cells

(A) mRNA of FACS-sorted peritoneal macrophages from ten SAMHD1-defi-cient, ten SAMHD1-deficient IFNAR1⁻/⁻, and ten control mice was analyzed by Illumina-based transcriptome sequencing. In total, 37 genes were upregulated at least 2-fold (p < 0.05) in SAMHD1-deficient mice compared with controls. All but two of these genes were known to be inducible by type I IFN (displayed as red dots). In the comparison of SAMHD1-deficient IFNAR1^{− /−} versus control cells, reduced transcript levels, rather than upregulation, were observed for most of these genes (black dots). (B) Transcript levels of five IFN-inducible genes found to be upregulated in the transcriptome analysis (A) were quantified in peritoneal macrophages from eight additional SAMHD1-deficient and 18 additional control mice by qRT-PCR. Fold upregulation for individual mutant samples compared with the mean of all control samples is displayed with colors identifying individual mice. (C) Transcript levels of three IFN-inducible genes were assessed in SAMHD1-deficient (n = 7) and control (n = 4) splenocytes, and in mutant (n = 5) and control (n = 4) E14.5 MEFs by qRT-PCR. Results are displayed as fold change for each mutant compared with the means of all controls. All genes were significantly upregulated (p < 0.04) in all mutant samples analyzed. (D) Transcript levels of three IFN-inducible genes in peritoneal macrophages from SAMHD1^Δ/Δ IFN-β-deficient (n = 8), SAMHD1^Δ/Δ IFN-βcompetent (n = 8), and SAMHD1^WT/WT IFN-β-deficient (n = 8) mice as determined by qRT-PCR. Results are displayed as fold change for each individual sample compared with the mean of all WT control samples (n = 8). See also Figure S3.

Figure 4. Spontaneous IFN Release Does Not Result in Autoimmune Disease in SAMHD1-Deficient Mice

(A) ANAs were detected by immunofluorescence analysis on murine fibroblasts. The significance of differences in ANA prevalence between groups was tested by chi-square test (Pearson) with Bonferroni correction. Autoantibodies occurred more frequently in Trex1⁻/⁻ mice (n = 19, mean age 10 weeks) compared with controls (Ctrl, p < 0.0001, n = 38, mean age 15 weeks) or SAMHD1-deficient mice (SAMHD1 KO, p < 0.005, n = 15 [this group comprised SAMHD1^KOF/KOF and SAMHD1^Δ/Δ mice, mean age 20 weeks]). No significant increase in ANA frequencies could be detected in the SAMHD1-deficient versus the control group. (B) Absence of inflammatory changes in heart and skin from SAMHD1-deficient mice. H&E-stained sections of heart and skin from one WT, one SAMHD1^KOF/KOF animal, and one diseased Trex1⁻/⁻ animal from our facility, representative of 13 SAMHD1^KOF/KOF mice analyzed (2 mice 16 weeks old, 11 older than 53 weeks) and five SAMHD1^Δ/Δ mice (3 mice 9–15 weeks old, 2 older than 43 weeks). See also Figure S4.