The ribonuclease activity of SAMHD1 is required for HIV-1 restriction

Nature Medicine, Journal Year: 2014, Volume and Issue: 20(8), P. 936 - 941

Published: July 16, 2014

Language: Английский

---

HIV accessory proteins versus host restriction factors

Current Opinion in Virology, Journal Year: 2013, Volume and Issue: 3(6), P. 692 - 699

Published: Nov. 15, 2013

Language: Английский

---

SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation

PLoS Genetics, Journal Year: 2015, Volume and Issue: 11(7), P. e1005367 - e1005367

Published: July 2, 2015

The SAM domain and HD containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses long interspersed element (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral synthesis by virtue dNTP triphosphohydrolase activity, may exploit a different mechanism to control LINE-1. Here, report new activity promoting cellular stress granule assembly, which correlates with increased phosphorylation eIF2α diminished eIF4A/eIF4G interaction. This enhances sequestration LINE-1 RNP granules consequent blockade retrotransposition. In support this action, depletion marker proteins G3BP1 or TIA1 abrogates formation overcomes inhibition Together, these data reveal for activating pathway.

Language: Английский

---

Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction

PLoS Pathogens, Journal Year: 2015, Volume and Issue: 11(10), P. e1005194 - e1005194

Published: Oct. 2, 2015

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural virological data demonstrating in addition allosteric activation activity, restriction correlates with the capacity form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes but has varied effects on vitro activity. phosphorylation also ablates tetramer formation without affecting steady-state kinetics. However phospho-SAMHD1 is unable catalyse turnover under conditions nucleotide depletion. Based our findings propose model for phosphorylation-dependent regulation dephosphorylation switches housekeeping found cycling cells high-activity stable tetrameric depletes maintains low levels dNTPs differentiated cells.

Language: Английский

---

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

Journal of Virology, Journal Year: 2014, Volume and Issue: 88(10), P. 5834 - 5844

Published: March 13, 2014

ABSTRACT Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of at threonine 592 (T592) cyclin-dependent kinase (CDK1) cyclin A2 impairs its HIV-1 restriction activity, but not dNTP hydrolase suggesting that depletion is sole mechanism SAMHD1-mediated restriction. Using coimmunoprecipitation mass spectrometry, we identified validated two additional host interacting with SAMHD1, namely, 2 (CDK2) S-phase kinase-associated protein (SKP2). We observed specifically interacted A2, B1, CDK1, CDK2. Given role these SAMHD1-interacting cell cycle progression, investigated regulation monocyte differentiation activation CD4 + T examined their effect on phosphorylation T592. Our results indicate primary T-cell regulate expression proteins. Furthermore, our suggest that, addition to CDK1 CDK2 phosphorylates T592 thereby regulates function. IMPORTANCE first triphosphohydrolase found mammalian cells. Previous studies suggested negatively activity. However, it unclear whether interacts other complex shares similar cellular partners. Here, identify five cycle-related interact including three previously unknown (CDK2, SKP2). demonstrate several play an important findings help define partners

Language: Английский

---

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

The EMBO Journal, Journal Year: 2017, Volume and Issue: 36(5), P. 604 - 616

Published: Jan. 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

Language: Английский

---

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

Nature Medicine, Journal Year: 2016, Volume and Issue: 22(10), P. 1072 - 1074

Published: Oct. 1, 2016

Language: Английский

---

SAMHD1 Functions and Human Diseases

Viruses, Journal Year: 2020, Volume and Issue: 12(4), P. 382 - 382

Published: March 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.

Language: Английский

---

Macrophages: Key Cellular Players in HIV Infection and Pathogenesis

Viruses, Journal Year: 2024, Volume and Issue: 16(2), P. 288 - 288

Published: Feb. 13, 2024

Although cells of the myeloid lineages, including tissue macrophages and conventional dendritic cells, were rapidly recognized, in addition to CD4+ T lymphocytes, as target HIV-1, their specific roles pathophysiology infection initially largely neglected. However, numerous studies performed over past decade, both vitro cell culture systems vivo monkey humanized mouse animal models, led growing evidence that play important direct indirect HIV-1 pathogenesis. It has been recently proposed are likely involved all stages pathogenesis, virus transmission dissemination, but above all, viral persistence through establishment, together with latently infected reservoirs many host tissues, major obstacle eradication people living HIV. Infected indeed found, very often multinucleated giant expressing antigens, almost lymphoid non-lymphoid tissues HIV-1-infected patients, where they can probably persist for long period time. In addition, also participate, directly targets or indirectly key regulators innate immunity inflammation, chronic inflammation associated clinical disorders observed HIV, even patients receiving effective antiretroviral therapy. The main objective this review is therefore summarize recent findings, revisit older data, regarding critical functions infection, found well during different

Language: Английский

---

CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain

Journal of Biological Chemistry, Journal Year: 2015, Volume and Issue: 290(21), P. 13279 - 13292

Published: April 7, 2015

Language: Английский

---