Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit

Nature Communications, Journal Year: 2018, Volume and Issue: 9(1)

Published: June 4, 2018

Abstract SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity regulated by T592 phosphorylation. Here, we show that dephosphorylation at controlled during the cell cycle, occurring M/G 1 transition proliferating cells. Using several complementary proteomics biochemical approaches, identify phosphatase PP2A-B55α responsible rendering antivirally active. specifically targeted holoenzymes mitotic exit, line with observations key exit mammalian Strikingly, as HeLa or activated primary CD4 + T enter G phase, pronounced reduction of RT products observed upon infection dependent on presence dephosphorylated SAMHD1. Moreover, PP2A controls pT592 level monocyte-derived macrophages (MDMs). Thus, holoenzyme regulator to switch

Language: Английский

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Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect

Scientific Reports, Journal Year: 2016, Volume and Issue: 6(1)

Published: Aug. 11, 2016

Abstract SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated is thought result from the depletion cellular pools, but it remains controversial whether dNTPase activity SAMHD1 sufficient for restriction. The ability regulated in cells by phosphorylation on T592. Phosphomimetic mutations T592 are not competent, appear intact their deplete dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization NMR-based enzymatic assays investigate impact phosphomimetic tetramerization vitro . We find that affect kinetics tetramer assembly disassembly, effects equilibrium insignificant. In contrast, Y146S/Y154S dimerization-defective mutant displays severe defect , indistinguishable WT its pools restrict HIV Our data suggest effect likely explain hypothesize subject additional regulatory mechanisms have yet been recapitulated

Language: Английский

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Phosphorylation of murine SAMHD1 regulates its antiretroviral activity

Retrovirology, Journal Year: 2015, Volume and Issue: 12(1)

Published: Dec. 1, 2015

Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes action have been extensively described for human SAMHD1, only little known about regulation mouse. Here, we characterize antiviral activity murine with help knockout mice to shed light on mechanism restriction validate mouse model use future infectivity studies.We found endogenous not HIV-1 but also MLV reporter virus infection at level reverse transcription primary myeloid Similar protein, regulated through phosphorylation threonine 603 limited nondividing Comparing susceptibility intracellular dNTP levels different cell types shows both functions are important determinants SAMHD1. In contrast, proposed RNase be less could detect any effect or incoming viral RNA.Our findings show blocks retroviral cycle-dependent phosphorylation. We mediated by mechanistically similar what making valuable tool influence vivo.

Language: Английский

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DNA damage induced by topoisomerase inhibitors activates SAMHD 1 and blocks HIV ‐1 infection of macrophages

The EMBO Journal, Journal Year: 2017, Volume and Issue: 37(1), P. 50 - 62

Published: Oct. 30, 2017

Article30 October 2017Open Access Source DataTransparent process DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages Petra Mlcochova Corresponding Author [email protected] orcid.org/0000-0001-6908-9363 Division Infection Immunity, UCL, London, UK Search for more papers this author Sarah J Caswell Macromolecular Structure Laboratory, The Francis Crick Institute, Ian A Taylor Greg Towers Ravindra K Gupta orcid.org/0000-0001-9751-1808 Africa Health Research Durban, KwaZulu Natal, South Information *,1, Caswell2, Taylor2, Towers1 Gupta1,3 1Division 2Macromolecular 3Africa *Corresponding author. Tel: +44 20 7679 2000; E-mail: EMBO Journal (2018)37:50-62https://doi.org/10.15252/embj.201796880 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract We report that inhibitors, etoposide (ETO), results in potent block human monocyte-derived (MDM). suppresses viral reverse transcription (RT) through depletion cellular dNTPs but is naturally switched off phosphorylation subpopulation MDM found G1-like state. was activated dephosphorylation following ETO treatment, along with loss expression MCM2 CDK1, reduction dNTP levels. Suppression occurred after completion synthesis, at step 2LTR circle provirus formation. ETO-induced completely rescued MDM. Concordantly, HIV-2 SIVsm encoding antagonist Vpx insensitive treatment. mechanism damage-induced blockade involved activation p53, p21, decrease CDK1 expression, dephosphorylation. Therefore, regulate HIV permissivity post-RT step, revealing which reservoir may be limited chemotherapeutic drugs. Synopsis Etoposide/camptothecin-mediated regulates macrophages. causes p53 leading transition from G0 state expression. In absence T592, resulting restriction infection. damage-mediated suppression occurs Introduction Retroviruses must transcribe their RNA into integrate nascent host genome order replicate (Skalka Katz, 2005; Lusic Siliciano, 2017). Increasing evidence suggests macrophage contributes infected cells persist prevent cure HIV/AIDS (Alexaki et al, 2008; Siliciano Greene, 2011; Watters 2013; Honeycutt 2016). Integration recognized as form damage, response (DDR) (Daniel 1999; Jackson Bartek, 2009) critical "gap repair" during integration process. Indeed, there increasing key DDR proteins are retroviral infection, specifically 2003; Ariumi DeHart Lau 2005). Furthermore, able restrict APOBEC3G SAMHD1, have been linked (Leonard Roberts Clifford 2014; Kretschmer 2015). deoxynucleotide triphosphohydrolase (Goldstone 2011), thought via levels insufficient Lahouassa 2012; Schmidt activity regulated CDK1/2-mediated amino acid T592 (Cribier White Antonucci 2016; Here, we link primary myeloid activation, type I interferon response. show dephosphorylation/activation. Activated mediates synthesis full-length DNA. Importantly, (ETO)-induced inhibition can fully abrogated depletion. Results induces post-reverse It has reported induce 2004). Moreover, certain reports suggest enhanced induction (Ebina Koyama 2013). also typically involves cell cycle arrest repair (Branzei Foiani, 2008). recently HDAC known and/or apoptosis, inhibit (MDM) (Mlcochova investigate impact inducing on employed unwinding dsDNA fundamental biological processes replication, transcription, chromatin remodelling, stabilizing DNA-single or -double breaks (Hsiang 1989; Gorczyca 1993). To test this, treated 5 μM 18 h confirm measured staining nuclear γH2AX 53BP1 (Fig 1A) uninfected cells. Further, pretreated (ETO) camptothecin (CTH), II, respectively. treatment titrations VSV-G pseudotyped GFP 48 later enumerating GFP-positive Both blocked dose-dependent manner 1B). then used most effective, non-cytotoxic, concentration (5 μM) 1C) measure its effect different viruses 1D). wild-type capsid mutants N74D P90A (known use alternative cofactors translocation) were equally sensitive inhibitors. However, SIVsm, encode Vpx, degrade drug viruses, adenovirus (AdV) herpes simplex virus (HSV), Semliki Forest (SFV), inhibition. Figure 1. Etoposide/Camptothecin-induced inhibits A. Monocyte-derived h. Cells stained foci, acquired analysed using automated cell-imaging system Hermes WiScan ImageJ. On average, 104 (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; paired t-test). Scale bars, 10 μm. B. concentrations (CTH) before presence ETO/CTH, each experiment recorded post-infection an ImageJ s.e.m.). C. CTH 66 distinguish between live dead LIVE/DEAD fixable Dead stain protocol. Percentages live/dead determined Addition 20% ethanol min positive control D. (wt) (N74D P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent AdV (AdV), (SFV) HSV-1. percentages normalized untreated ˜100% **P-value 0.01; ***P-value 0.001; (ns) non-significant, representative donor immunoblotting. E. GFP. 4, 0.001, F. tropic BaL. intracellular p24, percentage quantified FACS G–I. BaL isolated qPCR (G) Late RT products. AZT: AZT, reverse-transcriptase inhibitor, control. (H) circles. (I) Integrated copies DNA, Alu-Gag qPCR. J. (3, 6, 12 ffu/cell). K. h, ffu/cell) L. 6 data available online figure. Data 1 [embj201796880-sup-0002-SDataFig1.pdf] Download figure PowerPoint next investigated where life acts. endocytic entry route) CCR5 isolate 1E F). ETO-mediated suggesting independence sensitivity route entry. isolation determine efficiency 1G), formation (a entry) 1H; De Iaco 2013) Alu-gag PCR 1I). Surprisingly, not affected observed circles 1H) confirmed did affect products MOI (determined ffu/target cell), even though significantly decreased 1J K). any over time-course 1L). addition, although already very low MDM, detected dTTP dCTP EV1). Click here expand EV1. Quantification (ETO)Analysis unstimulated ETO-treated extracted quantification two donors. bar graph shows amounts (fmoles per 106 cells) indicated donor, without BD: below level detection. dependent lentiviruses (HIV-2 SIVsm), (Hrecka Laguette might responsible ETO/CTH ETO, assayed immunoblot 2A–C). phosphorylated allowing efficient confirming previous led reduced infectivity. As expected, inhibitory lost siRNA-mediated (SAMHD1 KD) 2A) SIVmac virus-like particles containing Vpx/Vpr (SIV VLP; Figs 2B EV2), dose titration KD EV3A) VLP-treated EV3B) infecting achieving equal EV3C D). rescue independent Vpr protein, evidenced VLP deleted (Figs 2C EV2) Vpx. inhibited consequently depletion, co-infection late 2D), 2E) 2F) under conditions FCS culture ETO. parallel samples post-viral challenge 2G). Critically, seen conclude mediated impacts both import integration, synthesis. 2. transfected pool (KD) siRNA 3 days later. co-infected VLP). VLP) del Vpx) Vpr). D–F. (F) products; circles; integrated (D) (E) G. 2 [embj201796880-sup-0003-SDataFig2.pdf] EV2. Schematic representation studySchematic packaging plasmids derived SIVmac251. prepared described Materials Methods. EV3. 5, noted increased nearly 20-fold increase mirrored proviral 2E bearing mutant Q76A rescues further probe (bearing WT/Vpr WT) mutant/Vpr maintains ability interact studies possibly because it cannot recruit DCAF1 (Srivastava Hrecka Reinhard 2014). cultured serum (low levels, dephosphorylated SAMHD1) (high 2017) 3A B). By contrast, had caused Q76A, degraded. EV4). (active) 3C) active manipulation degradation. 3. A, (WT)/Vpr Q76A) (A) differentiated instead FCS. (B) standard condition all experiments. See C–F. Q76A. (C) post-infection. [embj201796880-sup-0004-SDataFig3.pdf] EV4. Q76AAnalysis expressing Vpx(Q76A). introduction Vpx(Q76A) VLPs. 3C), 3D), 3E) 3F). ~3- 5-fold RT, This 3C–F). no substantial 3A–F). Interestingly, These antagonized being degraded T592. does trigger IFN Recent anti-microbial (Brzostek-Racine Hartlova shown mediate spontaneous release when mutated (Crow Manel, 2015) (Behrendt 2013), possibility could activate damage. innate immune triggering, cGAMP, product sensor cGAS (Sun Wu IFN-β tested specific interferon-stimulated gene (ISG) transcripts 4A). None ISG strongly elevated exposure immunofluorescence/translocation assay 5–20% IRF3-positive nuclei cGAMP LPS (positive controls), consistent responses. IRF3 translocation nucleus 2, 4B C), 4. μg/ml ng/ml performed selected genes TaqMan assays. Expression target GAPDH 100 (green) (blue). bars: promotes p21 general hypothesized entirely SAMHD1. demonstrated status Badia active, deactivated whether altered therefore same pathway, proportion G1 detection MCM2, marker expressed throughout G0. fact, thus indicative returning G0, non-permissive 5A). mapped pathway immunoblotting 5B–D). γH2AX, resulted Ser15 p27 protein. PARP cleavage 5B–D) suggested lack addition supported death viability/cell survival analysis 1C). Loss correlated No CDK2 5B C). obtained inhibitor 5D). p53/p21/CDK1 culminating 6). 5. CTH. lysed detect cycle/cell damage-associated proteins. band intensities panel CCD camera. Intensities protein bands intensity actin band. 0.01 protei

Language: Английский

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SAMHD1 and the innate immune response to cytosolic DNA during DNA replication

Current Opinion in Immunology, Journal Year: 2018, Volume and Issue: 56, P. 24 - 30

Published: Oct. 4, 2018

Language: Английский

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Constitutive Siglec-1 expression confers susceptibility to HIV-1 infection of human dendritic cell precursors

Proceedings of the National Academy of Sciences, Journal Year: 2019, Volume and Issue: 116(43), P. 21685 - 21693

Published: Oct. 7, 2019

The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population blood circulating DC precursors (pre-DCs). Whether this possesses specific functional features as compared to other subset upon pathogen encounter remained be evaluated. A unique feature pre-DCs among DCs is their constitutive expression viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not subsets, are susceptible infection HIV-1 in Siglec-1–dependent manner. Siglec-1 mediates pre-DC CCR5- and CXCR4-tropic strains. Infection further enhanced presence HIV-2/SIVmac Vpx, indicating does counteract restriction factors such SAMHD1. Instead, promotes attachment fusion particles. HIV-1–infected produce infectious particles accumulate intracellular compartments reminiscent virus-containing compartment macrophages. Pre-DC activation toll-like (TLR) ligands induces an antiviral state inhibits infection, remains replication-independent transfer activated primary T lymphocytes. Altogether, Siglec-1–mediated susceptibility constitutes might represent preferential relationship emerging type with viruses.

Language: Английский

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Ribonucleotide reductase inhibitors suppress SAMHD 1 ara‐ CTP ase activity enhancing cytarabine efficacy

EMBO Molecular Medicine, Journal Year: 2020, Volume and Issue: 12(3)

Published: Jan. 17, 2020

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well other haematological and lymphoid malignancies, but must be combined with chemotherapeutics to achieve cure. Yet, underlying mechanism dictating synergistic efficacy combination chemotherapy largely unknown. dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically ribonucleotide reductase (RNR), limits ara-C by hydrolysing active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors RNR, such gemcitabine hydroxyurea, overcome SAMHD1-mediated barrier in primary blasts mouse models AML, displaying SAMHD1-dependent synergy ara-C. We present evidence this is mediated pool imbalances leading allosteric reduction SAMHD1 ara-CTPase activity. Thus, constitutes a novel biomarker for therapies RNR immediate consequences clinical practice improve AML.

Language: Английский

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SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

Autoimmunity, Journal Year: 2018, Volume and Issue: 51(3), P. 96 - 110

Published: March 27, 2018

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a deoxynucleotide triphosphate (dNTP) hydrolase that plays an important role in the homeostatic balance of cellular dNTPs. Its emerging as effector innate immunity affirmed by mutations SAMHD1 gene cause severe autoimmune disease, Aicardi–Goutieres syndrome (AGS) are linked to cancer. Additionally, functions restriction factor for retroviruses, such HIV. Here, we review current biochemical biological properties enzyme including its structure, activity, regulation post-translational modifications context function. We outline open questions regarding biology whose answers will be understanding function regulator cell cycle progression, genomic integrity, autoimmunity.

Language: Английский

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With me or against me: Tumor suppressor and drug resistance activities of SAMHD1

Experimental Hematology, Journal Year: 2017, Volume and Issue: 52, P. 32 - 39

Published: May 12, 2017

Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) is a (deoxy)guanosine triphosphate (dGTP/GTP)-activated deoxyribonucleoside (dNTP) triphosphohydrolase involved in cellular dNTP homoeostasis. Mutations SAMHD1 have been associated with the hyperinflammatory disease Aicardi-Goutières syndrome (AGS). also limits cells' permissiveness to infection diverse viruses, including human immunodeficiency virus (HIV-1), controls endogenous retroviruses. Increasing evidence supports role of as tumor suppressor. However, can act resistance factor nucleoside-based chemotherapies by hydrolyzing their active metabolites, thereby reducing response various malignancies these anticancer drugs. Hence, informed cancer therapies must take into account ambiguous properties both an inhibitor uncontrolled proliferation limiting efficacy treatments. Here, we provide that double-edged sword for patients acute myelogenous leukemia (AML). Our time-dependent analyses The Cancer Genome Atlas (TCGA) AML cohort indicate high expression SAMHD1, even though it critically high-dose ara-C therapy, might be more favorable progression.

Language: Английский

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The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

Mobile DNA, Journal Year: 2018, Volume and Issue: 9(1)

Published: March 28, 2018

The restriction factor SAMHD1 regulates intracellular nucleotide level by degrading dNTPs and blocks the replication of retroviruses DNA viruses in non-cycling cells, like macrophages or dendritic cells. In patients, inactivating mutations samhd1 are associated with autoimmune disease Aicardi-Goutières Syndrome (AGS). accumulation nucleic acids derived from endogenous retroelements thriving absence has been discussed as potential trigger reaction. vitro, found to restrict retroelements, LINE-1 elements (L1). mechanism, however, which is still unclear. Here, we show that inhibits L1 other cycling By applying GFP- neomycin-based reporter assays anti-L1 activity regulated phosphorylation at threonine 592 (T592). Similar block HIV, cofactor binding site enzymatic active HD domain proofed be essential for elements. However, T592 did not correlate dNTP hydrolase 293T cells suggesting an alternative mechanism regulation. Interestingly, binds ORF2 protein this interaction phosphorylation. Together finding also our results suggest SAMHD1-mediated inhibition similar but identical HIV restriction. Our findings conclusively restricts vitro. important maintaining genome integrity support idea enhanced vivo, potentially triggering diseases AGS. analysis contributes better understanding activities antiviral defense metabolism. its against necessarily suggests might only function controlling autoimmunity.

Language: Английский

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