SAMHD1 shapes deoxynucleotide triphosphate homeostasis by interconnecting the depletion and biosynthesis of different dNTPs

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Янв. 17, 2025

SAMHD1 is a dNTPase that impedes replication of HIV-1 in myeloid cells and resting T lymphocytes. Here we elucidate the substrate activation mechanism SAMHD1, which involves dNTP binding at allosteric sites transient tetramerization. Our findings reveal tetramerization alone insufficient to promote hydrolysis; instead, requires an inactive tetrameric intermediate with partially occupied sites. The equilibrium between active states regulates activity, driven by dissociation additional ligands preassembled tetramer. Furthermore, catalytic efficiency, but not specificity, modulated identity dNTPs occupying We show how this regulation shapes deoxynucleotide homeostasis balancing production SAMHD1-catalyzed depletion. Notably, exhibits distinct functionality, term facilitated depletion, whereby increased biosynthesis certain enhances depletion others. regulatory relationship different sheds light on emerging role biology implications for HIV/AIDS, innate antiviral immunity, cell disorders, telomere maintenance therapeutic efficacy nucleoside analogs.

Язык: Английский

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SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways

Proceedings of the National Academy of Sciences, Год журнала: 2018, Номер 115(16)

Опубликована: Апрель 2, 2018

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN inflammatory responses viral infections, although functions mechanisms in modulating innate immunity remain unclear. Here, we show that suppresses immune infections stimuli inhibiting nuclear factor-κB (NF-κB) activation type I interferon (IFN-I) induction. Compared with control cells, infection SAMHD1-silenced human monocytic cells or primary macrophages Sendai virus (SeV) HIV-1, treatment stimuli, induces significantly higher levels NF-κB IFN-I Exogenous expression reconstitution knockout induction SeV stimuli. Mechanistically, inhibits interacting NF-κB1/2 phosphorylation inhibitory IκBα. also interacts inhibitor-κB kinase ε (IKKε) regulatory factor 7 (IRF7), leading suppression pathway IKKε-mediated IRF7 phosphorylation. Interactions endogenous proteins were validated macrophages. Comparing splenocytes from heterozygous mice, further confirmed SAMHD1-mediated activation, suggesting an evolutionarily conserved property SAMHD1. Our findings reveal down-regulating highlighting importance antiviral immunity.

Язык: Английский

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A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

The EMBO Journal, Год журнала: 2017, Номер 36(5), С. 604 - 616

Опубликована: Янв. 25, 2017

Article25 January 2017Open Access Source DataTransparent process A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages Petra Mlcochova Division of Infection and Immunity, University College London, UK Search for more papers by this author Katherine Sutherland Sarah Watters Cosetta Bertoli MRC Laboratory Molecular Cell Biology, Rob AM de Bruin Jan Rehwinkel Medical Research Council Human Immunology Unit, Radcliffe Department Medicine, Weatherall Institute Oxford, Stuart J Neil Immunology, Inflammatory Disease, King's College, Gina M Lenzi Pediatrics, Center Drug Discovery, Emory School Atlanta, GA, USA Baek Kim Asim Khwaja Haematology, UCL, Matthew C Gage Christiana Georgiou Alexandra Chittka Simon Yona Mahdad Noursadeghi Greg Towers Ravindra K Gupta Corresponding Author [email protected] orcid.org/0000-0001-9751-1808 Africa Health Institute, KwaZulu Natal, South Information Mlcochova1, Sutherland1, Watters1, Bertoli2, Bruin2, Rehwinkel3, Neil4, Lenzi5, Kim5, Khwaja6, Gage7, Georgiou7, Chittka7, Yona7, Noursadeghi1, Towers1 *,1,8 1Division 2MRC 3Medical 4Division 5Department 6Research 7Division 8Africa *Corresponding author. Tel: +44 20 7679 2000; E-mail: The EMBO Journal (2017)36:604-616https://doi.org/10.15252/embj.201696025 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract An unresolved question is how achieves efficient replication terminally differentiated despite factor SAMHD1. We reveal inducible changes expression cell cycle-associated proteins MCM2 cyclins A, E, D1/D3 macrophages, without evidence DNA synthesis or mitosis. These are induced activation Raf/MEK/ERK kinase cascade, culminating upregulation CDK1 with subsequent T592 phosphorylation deactivation its antiviral activity. HIV infection limited these phase at single-cell level. Depletion decouples association between proteins, becoming highly susceptible HIV-1. observe both embryo-derived monocyte-derived tissue-resident frequencies approaching 20%, suggesting sustain vivo. Finally, we SAMHD1-dependent antiretroviral activity histone deacetylase inhibitors acting via p53 activation. data provide basis host-directed therapeutic approaches aimed limiting burden that may contribute curative interventions. Synopsis Here, show found G0- phases, latter permissive due canonical pathway which deactivates CDK1-mediated T592. Therefore, does not need encode antagonist. Macrophages transition classical G0 quiescent state, representing up 20% tissue macrophages. G0–G1 regulated pathway, associated increased protein, permissivity window infection. Histone possess Introduction SAMHD1, deoxynucleotide-triphosphate (dNTP) hydrolase, restricts reverse transcription (RT) through decreasing levels dNTPs (Goldstone et al, 2011; Lahouassa 2012; Schmidt 2015). position mediated cyclin-dependent kinases CDK1/2 (Cribier 2013; White 2013) actively dividing cells impairs dNTP hydrolase viral occur Arnold Some lentiviruses have evolved countermeasures against SAMHD1; example, HIV-2/SIVsm lineage encodes Vpx protein degrades otherwise SAMHD1-positive target (Kaushik 2009; Hrecka Laguette 2011). How pandemic strains achieve vivo Vpx-like has remained significant our understanding tropism pathogenesis (Watters 2013). dynamic non-dividing do culminate dependent on mitogen/growth factor-activated signalling sufficient deactivate potent Moreover, two distinct populations mice express profile, providing only an explanation ability high but also offering vital innate immune cells. Results Terminally stimulated enter observed culture human (MDM) foetal calf serum/FCS (stimulated cells), as opposed serum/HS (unstimulated led increase (Figs 1A–C EV1A–E). As expected, there was donor variation absolute (Fig EV1B). under stimulating conditions single-round VSV-G-pseudotyped virus 1A EV1A B) full-length infectious molecular clones 1B C, EV1C D), macrophage tropic viruses (BaL, YU-2), clinical isolates D) capsid mutations known alter interactions cyclophilin CPSF6 leading altered transcription, retargeted integration triggering sensing EV1E). enhancement post-entry step EV1F). effect FCS lost when charcoal-stripped used boiled serum/foetal serum mixture (1:1) used, existence heat stable stimulatory rather than inhibitory HS EV1A). Figure 1. Transitioning from MDM were cultured RPMI complemented MCSF 10% (unstim) (stim). infected expressing GFP, percentage quantified 48 h post-infection FACS (n = 3, mean ± s.e.m.; **P-value ≤ 0.01, unpaired t-test). BaL isolated 18 qPCR late RT products *P-value 0.05, Spreading MDM. Cells BaL, stained intracellular p24 FACS. Principal component analysis macrophage-associated transcripts compare relative clustering unstimulated range primary cells/cell lines. Diagram cellular functions genes (−log2(P-value) > 2) transcriptional compared Immunoblot expressed Star indicates non-specific band. This Western blot quantification Donor 1 Fig 2A. same blots 2A allow comparison different well Uninfected exposed VSV-G GFP (HIV-1 exposed) MCM2, Geminin EdU incorporation (EdU added 50 prior analysis). On average, 104 each experiment recorded analysed using Hermes WiScan cell-imaging system ImageJ. cycle markers shown. quantitation content flow cytometry. Cycling THP-1 labelled propidium iodide (PI) CFSE loaded 4 days determine division/proliferation available online figure. Data [embj201696025-sup-0004-SDataFig1.pdf] Download figure PowerPoint Click here expand EV1. Enhanced susceptibility 3 additional (unstim), (stim), (CS), 5 min filtrated 0.45-μm filter (FCS boil), 1:1 mix (unstim:stim 1:1), (unstim:FCS boil 1:1) then HIV-1-expressing GFP. detected 12 donors (D1-D12) GFP; Single round panel (macrophage viruses: YU2; isolates: CH77, CH58, ZM247, WITO). numbers light microscopy. titres released determined indicator HeLa TZM-bl. Culture supernatants BaL-infected harvest day 2, 6 9 post-infection, filtered infect TZM-bl Luciferase measured 24 post-infection. (wt HIV-1; mutants P90A, N74D, G89V), All displayed similar (which normalised ˜100%). equal amounts (50 ng) BlaM-Vpr-containing h. CCF2/AM dye, fusion events cytometry BD LSR Fortessa gated 10,000 information: Graphs average n ≥ 0.05; 0.01; ***P-value 0.001, t-test. differential effects suggested approach uncovering mechanisms regulating transcriptomes cells, UNSTIM) STIM) aiming discover pathways higher permissivity. Comparison profiles predefined gene signature discriminates other types (Tomlinson 2012) clearly shows cluster together closely related myeloid 1D). they EV2). However, use ingenuity evaluate revealed enrichment number molecular/cellular regulation, growth proliferation, death survival 1E). top enriched encoding involved regulation 1F EV3A, Table EV1). observations validated level 1G). Stimulated showed D-type D1 D3, accumulate progress G1 (Baldin 1993; Sherr, 1993, 1996). Cyclin D2 below detectable E2F6 Geminin—all during entry (Coverley 2002; Fragkos Of note, CDK1—a key player progression—was upregulated along (minichromosome maintenance complex 1G H), origin licensing (but G0) 1996; Su O'Farrell, 1997; Tsuruga Musahl 1998; Stoeber 1998, 2001; Williams 1998). Importantly, p27 reduced following stimulation inhibitor decreases after re-entry (Sherr Roberts, 1999). EV2. Expression surface unaffected B. (CD68, CD14) M1 M2 (CD163, CD80, CD86 CD40) EV3. can be manipulated Ingenuity interaction nuclear demonstrates single dominant interacting proteins. Example acquisition system, automated microscopic platform. nuclei, active Click-iT® Alexa Fluor® 488 Imaging Kit. Scale bars: 10 μm. treated μM detect EdU-positive Quantification phases PI labelling. 7 changed into medium (10% FCS) days. CDK4/6 Palbociclib (1 μM) before virus; VLP-vpx time lysed immunoblotting. ImageJ s.e.m.). further absent G0/quiescent/terminally (i) all (MCM2) (Masai 2010), (ii) S G2/M (Geminin) (Fragkos 2015) (iii) specific therefore marker incorporation) 1H). platform EV3B) versus positive fivefold 10-fold S-G2-M Together low over 50-h period, suggest re-entered majority did S, 1H EV3C). confirmed divide staining 1I J, EV3D). able modulate measurable division synthesis. early consistent Transition regulate Given well-described Pauls 2014; Yan 2015), hypothesised might spontaneously To test this, examined knowing phosphorylates capacity restrict Welbourn raised pSAMHD1-T592 MDM, CDK2, CDK4 CDK6 2A). threefold fourfold EV3E), reported (Lahouassa 2012). Furthermore, exogenously degradation co-infection SIVmac virus-like particles bearing (VLP-vpx) 2B), depletion siRNA transfection 2C) infectivity specifically no change where phosphorylated thus already inactive 2. Bidirectional transitions shape SAMHD1-mediated A. three (D1, D2, D3) immunoblotting CDK order facilitate co-infected containing vpx (VLP-vpx). representative (ns) non-significant, C. transfected control pool siRNAs later D. Experimental model bidirectional G0–G1-like transitions. follows: (stim) described 4; [stim (day 3)] grown days; [unstim (3 days)] condition non-stimulating remaining E. BaL. CDKs, Graph example s.e.m. F. Proposed G, H. Unstimulated (G) (H) RAF (2 μM), B-RAF MEK1/2 (AS-703026, JAK 1–3 GSK3 PIM s.e.m., calculated triplicates, I. MEK/ERK (U0126, indicated Percentage non-significant; 2 [embj201696025-sup-0005-SDataFig2.pdf] probe reversibility unstimulating FCS, vice versa 2D E). Non-stimulating onwards 3), E] expressions (indicative returning state), decreased Conversely, EV3F) augmented MCM2/CDK1 re-entering state) phosphorylation. No CDK2 2E). reversible explore mapped responsible well-characterised 2F–I). identified involvement cascade 2F–I EV3G). B-Raf PLX4032 (active V600E mutant cancer cells) negative demonstrate specificity Raf inhibition 2G H). pharmacologically block putative signal activated MEK/ERK, U0126, reasoning would lead suppression manner 2F). Indeed, substantially inhibited loss correlated dephosphorylation, downregulation 2I). Critically, 2I) completely rescued U0126. inhibiting downstream illustrate, first time, regulates preferential targets next employed high-throughput co-localisation analysis, measure progression 3). (added infection) monitor 3A–E). non-stimulatory 3A–C G–J). illustrated expression, Stimulation simply MCM2-positive 3B, explains cultures 1A. even though EV4A), population synthesising minority (< 7%) statistic

Язык: Английский

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From APOBEC to ZAP: Diverse mechanisms used by cellular restriction factors to inhibit virus infections

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Год журнала: 2018, Номер 1866(3), С. 382 - 394

Опубликована: Окт. 2, 2018

Язык: Английский

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SAMHD1 Functions and Human Diseases

Viruses, Год журнала: 2020, Номер 12(4), С. 382 - 382

Опубликована: Март 31, 2020

Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells a variety viral pathogens. While process dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) thymidine kinase (TK), has been extensively investigated, negative regulatory mechanism pools was recently found to involve sterile alpha motif (SAM) domain histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, triphosphohydrolase activity SAMHD1 degrades dNTPs into their 2'-deoxynucleoside (dN) subparts, steadily depleting intercellular pools. The differential expression levels activation states various cell types contributes unique that either aid (i.e., dividing T cells) or restrict nondividing macrophages) consumes dNTPs. Genetic mutations induce rare inflammatory encephalopathy called Aicardi-Goutières syndrome (AGS), which phenotypically resembles infection. Recent publications have identified diverse roles double-stranded break repair, genome stability, stress response through interferon signaling. Finally, series were also reported cancer while why is mutated these remains investigated. Here, we reviewed studies begun illuminating highly virology, immunology, biology.

Язык: Английский

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Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

AIDS Research and Human Retroviruses, Год журнала: 2017, Номер 33(11), С. 1155 - 1165

Опубликована: Май 10, 2017

Macrophages are a target of human immunodeficiency virus type 1 (HIV-1) infection and may serve as an important reservoir the in body, particularly after depletion CD4+ T cells HIV/AIDS. Recently, sterile alpha motif histidine/aspartic acid domain-containing protein (SAMHD1) was identified major restriction factor HIV-1 myeloid cells. SAMHD1 is targeted for proteolytic degradation by Vpx, viral encoded HIV-2 many simian viruses but not HIV-1. In this study, we assessed vitro differentiated macrophages freshly isolated from lungs, abdomen, brain. We found that spread cultured monocyte-derived were highly limited Vpx largely relieved to initial infection, expected. observed nearly identical profiles peripheral blood monocytes, well lung (alveolar) abdominal (peritoneal) macrophages. contrast, under same conditions, primary brain (microglia) susceptible despite levels endogenous comparable other macrophage populations. Addition further increased conditions limiting input, robust whether or depleted. These results suggest peripherally circulating effectively restricted SAMHD1; however, microglia expression. data explain long-standing observation often detected brain, seldom tissues body.

Язык: Английский

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Conserved Herpesvirus Protein Kinases Target SAMHD1 to Facilitate Virus Replication

Cell Reports, Год журнала: 2019, Номер 28(2), С. 449 - 459.e5

Опубликована: Июль 1, 2019

Highlights•SAMHD1 depletion facilitates EBV lytic replication•EBV protein kinase BGLF4 directly phosphorylates SAMHD1•BGLF4 phosphorylation of SAMHD1 inhibits its dCTPase and dTTPase activity•SAMHD1 is targeted by the conserved herpesvirus kinasesSummaryTo ensure a successful infection, herpesviruses have developed elegant strategies to counterbalance host anti-viral responses. Sterile alpha motif HD domain 1 (SAMHD1) was recently identified as an intrinsic restriction factor for variety viruses. Aside from HIV-2 related simian immunodeficiency virus (SIV) Vpx proteins, direct viral countermeasures against remain unknown. Using Epstein-Barr (EBV) primary model, we discover that SAMHD1-mediated antagonized BGLF4, member kinases encoded all herpesviruses. Mechanistically, find thereby deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity. We further demonstrate targeting common feature shared beta- gamma-herpesviruses. Together, our findings uncover immune evasion mechanism whereby exploit thwart defenses.Graphical abstract

Язык: Английский

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SAMHD1 protects cancer cells from various nucleoside-based antimetabolites

Cell Cycle, Год журнала: 2017, Номер 16(11), С. 1029 - 1038

Опубликована: Апрель 24, 2017

Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), most important drug AML treatment. Ara-C intracellularly converted by canonical dNTP synthesis pathway ara-CTP, which serves as substrate but not an allosteric activator SAMHD1. Using mouse model, show here wild type catalytically inactive SAMHD1 reduces ara-C treatment efficacy vivo. Expanding clinically relevant substrates SAMHD1, demonstrate THP-1 CRISPR/Cas9 lacking functional gene showed increased sensitivity antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, trifluridine. Within this Extra View, discuss build upon both these our previously reported findings, propose likely active against variety nucleoside analog present anti-cancer chemotherapies. Thus, may constitute promising target improve wide range therapies for hematological non-haematological malignancies.

Язык: Английский

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Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

Scientific Reports, Год журнала: 2016, Номер 6(1)

Опубликована: Май 27, 2016

Abstract Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV We demonstrated that silencing SAMHD1 in hepatic cells increased replication, while overexpression had opposite effect. significantly affected levels extracellular viral DNA as well intracellular reverse transcription products, without affecting RNAs or cccDNA. mutations interfere with dNTPase activity (D137N) catalytic center histidine-aspartate (HD) domain (D311A), phospho-mimetic mutation (T592E), abrogated inhibitory activity. In contrast, diminishing potential RNase but not (Q548A) disabling phosphorylation (T592A) did affect antiviral Moreover, by was rescued addition deoxynucleosides. Although infection directly protein level upregulated dATPs. Interestingly, dephosphorylated, thus potentially antiviral-active state, primary human hepatocytes. Furthermore, type I II interferons cells. These results suggest is relevant restricts through its

Язык: Английский

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Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1

Virology, Год журнала: 2015, Номер 488, С. 271 - 277

Опубликована: Дек. 4, 2015

Язык: Английский

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