SAMHD1 restricts the deoxyguanosine triphosphate pool contributing to telomere stability in telomerase-positive cells.

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.

SAMHD1-deficient fibroblasts from Aicardi-Goutières Syndrome patients can escape senescence and accumulate mutations.

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.

Analysis on biological functions of restriction factor SAMHD1 of primates / 吉林大学学报(医学版)

Objective: To discuss the antiviral effect, the inhibitory effect on LINE-1 retrotransposon activity and the redection of interferon production signal pathway of restriction factor SAMHD1 of the primates, and to provide the basis for further study of the SAMHD1 of the primates. Methods: The U937 cells stably expressing the SAMHD1 of primates were established; the U937-control cells established with pLVX -puro were used as negative control group, and the U937-SAMHD1 cells stably expressing the SAMHD1 protein of the different primates were used as experimental groups; the cells were treated with PMA to induce cell differentiation. The virus infection rates of HIV-1 in the cells in various groups were determined by flow cytometry. The HEK293T cells transfected with the expression plasmid of SAMDH1 were used as control group, and the cells co-transfected with the SAMHD1 and HIV-2/SIV Vpx expression plasmids were used as experimental groups. The cells were obtained 48 h after transfection, and the expression levels of SAMHD1 protein were determined by Western blotting method. The intracellular location of SAMHD1 protein was determined by immunofluorescence. The HEK293T cells transfected with LINE-1-GFP report plasmid were used as control group, and the cells co-transfected with LINE-1-GFP and SAMHD1 expression plasmids were used as experimental groups. The rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) were determined by flow cytometry. The HEK293T cells transfected with IFN- Luc report plasmid were used as control group, and the cells co-transfected with IFN-Luc and pSAMHDl expression plasmids were used as experimental groups. The expression levels of luciferase in HEK293T cells were determined by chemiluminescence instrument. Results: Compared with negative control group, the virus infection rates of HIV-1 in experimental groups with stable expression of SAMHD1 in the primates were significantly decreased (P<0. 01). Compared with control group, the expression levels of SAMHD1 protein of the primates in experimental groups were decreased (P<0. 05 or P<0. 01). The immunofluorescence results showed that the SAMHD1 protein of the primates was localized in the nucleus. Compared with control group, the rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) in experimental groups were significantly decreased (P< 0.05 or P<0.01). Compared with control group, the expression levels of luciferase in the HEK293T cells in experimental groups were significantly decreased (P<0. 05). Conclusion: The SAMHD1 protein of the different primates can resist the HIV-1 infection, inhibit the LINE-1 retrotransposon and antagonize the IFN production by natural immune system.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592.

SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication.

The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response.

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigen-presenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4+ T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1's role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.