SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis.

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes.

Spatial resolution of HIV-1 post-entry steps in resting CD4 T cells.

Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.

Molecular dynamics investigation of DNA fragments bound to the anti-HIV protein SAMHD1 reveals alterations in allosteric communications.

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Impaired influenza A virus replication by the host restriction factor SAMHD1 which inhibited by PA-mediated dephosphorylation of the host transcription factor IRF3.

BACKGROUND: Influenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV. METHODS: To investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication. RESULTS: In this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1's ability to restrict H5N1 is related to phosphorylation at 592-tyrosine. CONCLUSIONS: In conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.

Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Activation of STING by SAMHD1 Deficiency Promotes PANoptosis and Enhances Efficacy of PD-L1 Blockade in Diffuse Large B-cell Lymphoma.

Genomic instability is a significant driver of cancer. As the sensor of cytosolic DNA, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a critical role in regulating anti-tumor immunity and cell death. However, the role and regulatory mechanisms of STING in diffuse large B-cell lymphoma (DLBCL) are still undefined. In this study, we reported that sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency induced STING expression and inhibited tumor growth in DLBCL. High level of SAMHD1 was associated with poor prognosis in DLBCL patients. Down-regulation of SAMHD1 inhibited DLBCL cell proliferation both in vitro and in vivo. Moreover, we found that SAMHD1 deficiency induced DNA damage and promoted the expression of DNA damage adaptor STING. STING overexpression promoted the formation of Caspase 8/RIPK3/ASC, further leading to MLKL phosphorylation, Caspase 3 cleavage, and GSDME cleavage. Up-regulation of necroptotic, apoptotic, and pyroptotic effectors indicated STING-mediated PANoptosis. Finally, we demonstrated that the STING agonist, DMXAA, enhanced the efficacy of a PD-L1 inhibitor in DLBCL. Our findings highlight the important role of STING-mediated PANoptosis in restricting DLBCL progression and provide a potential strategy for enhancing the efficacy of immune checkpoint inhibitor agents in DLBCL.

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.

IMPORTANCE: We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

A nuclear orthologue of the dNTP triphosphohydrolase SAMHD1 controls dNTP homeostasis and genomic stability in Trypanosoma brucei.

Maintenance of dNTPs pools in Trypanosoma brucei is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and de novo pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of T. brucei. Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of TbHD82 null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.

SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1 cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1 cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages.

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Protein oxidation increases SAMHD1 binding ssDNA via its regulatory site.

SAMHD1 dNTP hydrolase activity places it at the crossroad of several important biological pathways, such as viral restriction, cell cycle regulation, and innate immunity. Recently, a dNTPase independent function for SAMHD1 in homologous recombination (HR) of DNA double-strand breaks has been identified. SAMHD1 function and activity is regulated by several post-translational modifications, including protein oxidation. Here, we showed that oxidation of SAMHD1 increases ssDNA binding affinity and occurs in a cell cycle-dependent manner during S phase consistent with a role in HR. We determined the structure of oxidized SAMHD1 in complex with ssDNA. The enzyme binds ssDNA at the regulatory sites at the dimer interface. We propose a mechanism that oxidation of SAMHD1 acts as a functional switch to toggle between dNTPase activity and DNA binding.

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells.

BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells.

Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.

Purified recombinant lentiviral Vpx proteins maintain their SAMHD1 degradation efficiency in resting CD4+ T cells.

Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4+ T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4+ T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.

The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKß) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/ß/γ via interaction with IKKα and IKKß, thus inhibiting subsequent phosphorylation of IκBα in monocytic THP-1 cells and differentiated nondividing THP-1 cells. We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKß, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/ß/γ in Sendai virus-infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKß in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKß directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKß and that the kinase domain of IKKα and the ubiquitin-like domain of IKKß are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKß. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.

Thymidine nucleotide metabolism controls human telomere length.

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.

Interferon-inducible SAMHD1 restricts viral replication through downregulation of lipid synthesis.

Background: Type I interferon (IFN) inhibits virus infection through multiple processes. Recent evidence indicates that IFN carries out its antiviral activity through readjusting of the cellular metabolism. The sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1), as an interferon-stimulated gene (ISG), has been reported to inhibit a number of retroviruses and DNA viruses, by depleting dNTPs indispensable for viral DNA replication. Here we report a new antiviral activity of SAMHD1 against RNA viruses including HCV and some other flaviviruses infection. Methods: Multiple cellular and molecular biological technologies have been used to detect virus infection, replication and variation of intracellular proteins, including western blotting, qRT-PCR, Gene silencing, immunofluorescence, etc. Besides, microarray gene chip technology was applied to analyze the effects of SAMHD1 overexpression on total expressed genes. Results: Our data show that SAMHD1 down-regulates the expression of genes related to lipid bio-metabolic pathway, accompanied with impaired lipid droplets (LDs) formation, two events important for flaviviruses infection. Mechanic study reveals that SAMHD1 mainly targets on HCV RNA replication, resulting in a broad inhibitory effect on the infectivity of flaviviruses. The C-terminal domain of SAMHD1 is showed to determine its antiviral function, which is regulated by the phosphorylation of T592. Restored lipid level by overexpression of SREBP1 or supplement with LDs counteracts with the antiviral activity of SAMHD1, providing evidence supporting the role of SAMHD1-mediated down-regulation of lipid synthesis in its function to inhibit viral infection. Conclusion: SAMHD1 plays an important role in IFN-mediated blockade of flaviviruses infection through targeting lipid bio-metabolic pathway.