Prof Jan Rehwinkel

Research Area: Immunology
Scientific Themes: Immunology and Microbiology, Infection & Tropical Medicine
Keywords: RIG-I-like receptors, influenza A virus, Aicardi-Goutieres syndrome and SAMHD1
Web Links:
Figure 1. Induction and effects of IFN during virus infection.

Virus infection delivers nucleic acids into the cytosol or endosomal compartment. Innate nucleic acid sensors including toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and cytosolic DNA receptors (CDRs) detect these DNAs and RNAs and then trigger a signal transduction cascade that induces IFN. Adaptor proteins, kinases and transcription factors mediate signalling. Note that additional proteins have been implicated and that the figure only shows some selected key components. IFN signals via IFNAR resulting in the induction of interferon-stimulated genes (ISGs) that have direct and indirect anti-viral effects.

Figure 1. Induction and effects of IFN during virus infection. Virus infection delivers nucleic ...

Figure 2 RIG-I recognizes the 5’-PPP-bearing influenza A virus RNA genome.

A schematic representation of the flu life cycle is shown. Flu has a segmented genome consisting of eight negative sense RNA molecules carrying 5’-PPP groups. Virus particles enter cells via endocytosis, followed by uncoating and nuclear replication of the viral genome (vRNA). This involves a positive sense anti-genome intermediate (cRNA). The viral genome is also transcribed in the nucleus into capped viral messenger RNAs (mRNAs) that are exported to the cytoplasm and translated. Viral proteins and replicated viral genomes assemble progeny virus particles that bud from the cell surface. IFN induction during flu infection is mediated by RIG-I recognition of progeny viral genomes and signalling via the mitochondrial adaptor protein MAVS. CARD, caspase activation and recruitment domain; Helicase, DExD/H-box RNA helicase domain; RD/CTD, repressive domain, also known as C-terminal domain; TM, transmembrane domain.

Figure 2 RIG-I recognizes the 5’-PPP-bearing influenza A virus RNA genome. A schematic ...

NUCLEIC ACID SENSING BY INNATE IMMUNE RECEPTORS

The innate immune response is critical for successful host defence against virus infection. Cell-intrinsic mechanisms detect virus presence and signal for the induction of innate response genes such a type I interferons (IFNs). Nucleic acids are often a molecular signature of virus infection and are recognised by innate receptors including toll-like receptors, RIG-I-like receptors and cytosolic DNA receptors (Figure 1). In addition to their protective role in infectious disease, some of these receptors have also been implicated in inflammatory conditions.

Our research dissects the molecular biology of activation and regulation of cytosolic nucleic acid sensors, during both virus infection and in inflammatory diseases. Previously, we identified the RNA recognized by RIG-I in cells infected with negative strand RNA viruses such as influenza A virus as progeny viral RNA genomes (Figure 2). Interestingly, these genomes bear a triphosphate moiety on the 5’-end that is essential for RIG-I activation and IFN induction. Such 5’-PPP-groups are absent from cellular RNA in the cytoplasm, providing an explanation for the specific triggering of RIG-I in virus-infected cells.

Currently, we are developing several complementary projects focused on three key questions: (1) What are the mechanisms of activation of the RNA binding receptors RIG-I and MDA5, and of cytosolic DNA sensors, during virus infection? (2) How is the expression and sub-cellular localization of nucleic acid binding receptors regulated? (3) What is the molecular basis of genetic disorders characterised by chronic type I IFN production in the absence of virus infection? This work at the intersection of molecular biology and immunology will provide important insights into activation of the innate immune system by nucleic acids. Our findings will have major implications for virus infection, inflammatory and autoimmune diseases and the development of vaccine adjuvants. Our work is funded by the UK Medical Research Council and by a Wellcome Trust New Investigator Grant.

Name Department Institution Country
Dr Anna Schuh Nuffield Division of Clinical Laboratory Sciences Oxford University, John Radcliffe Hospital United Kingdom
Prof Alexander (Hal) Drakesmith Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Graham Ogg Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor Alison Simmons Experimental Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Kerstin Luhn Investigative Medicine Division Oxford University,
Andrew Jackson Medical Research Council Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh United Kingdom
Yanick Crow Manchester Academic Health Science Centre, University of Manchester, Genetic Medicine, Manchester United Kingdom
Caetano Reis e Sousa Immunobiology Laboratory, Cancer Research UK London Research Institute United Kingdom
Marco Colonna Department of Pathology and Immunology, Washington University School of Medicine, St. Louis United States
Glück S, Guey B, Gulen MF, Wolter K, Kang TW, Schmacke NA, Bridgeman A, Rehwinkel J, Zender L, Ablasser A. 2017. Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence. Nat Cell Biol, 19 (9), pp. 1061-1070. | Show Abstract | Read more

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

Maelfait J, Liverpool L, Bridgeman A, Ragan KB, Upton JW, Rehwinkel J. 2017. Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis. EMBO J, 36 (17), pp. 2529-2543. | Show Abstract | Read more

Nucleic acids are potent triggers for innate immunity. Double-stranded DNA and RNA adopt different helical conformations, including the unusual Z-conformation. Z-DNA/RNA is recognised by Z-binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM-1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z-DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1-ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA These observations show that Z-RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.

Maelfait J, Rehwinkel J. 2017. RECONsidering Sensing of Cyclic Dinucleotides. Immunity, 46 (3), pp. 337-339. | Show Abstract | Read more

Detection of cyclic dinucleotides (cdNs) by the STING pathway potently triggers the antiviral response. McFarland et al. now show that the mouse oxidoreductase RECON acts as a sensor for some bacterial cdNs, modulating innate signaling in a manner independent of STING to promote an antibacterial state.

Mlcochova P, Sutherland KA, Watters SA, Bertoli C, de Bruin RA, Rehwinkel J, Neil SJ, Lenzi GM, Kim B, Khwaja A et al. 2017. A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages. EMBO J, 36 (5), pp. 604-616. | Show Abstract | Read more

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.

Donald CL, Brennan B, Cumberworth SL, Rezelj VV, Clark JJ, Cordeiro MT, Freitas de Oliveira França R, Pena LJ, Wilkie GS, Da Silva Filipe A et al. 2016. Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil. PLoS Negl Trop Dis, 10 (10), pp. e0005048. | Show Abstract | Read more

BACKGROUND: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. CONCLUSIONS/SIGNIFICANCE: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

Rehwinkel J. 2016. Is anti-viral defence the evolutionary origin of mRNA turnover? (Comment on DOI 10.1002/bies.201600100). Bioessays, 38 (9), pp. 817. | Read more

Maelfait J, Bridgeman A, Benlahrech A, Cursi C, Rehwinkel J. 2016. Restriction by SAMHD1 Limits cGAS/STING-Dependent Innate and Adaptive Immune Responses to HIV-1. Cell Rep, 16 (6), pp. 1492-1501. | Show Abstract | Read more

SAMHD1 is a restriction factor for HIV-1 infection. SAMHD1 mutations cause the autoinflammatory Aicardi-Goutières syndrome that is characterized by chronic type I interferon (IFN) secretion. We show that the spontaneous IFN response in SAMHD1-deficient cells and mice requires the cGAS/STING cytosolic DNA-sensing pathway. We provide genetic evidence that cell-autonomous control of lentivirus infection in myeloid cells by SAMHD1 limits virus-induced production of IFNs and the induction of co-stimulatory markers. This program of myeloid cell activation required reverse transcription, cGAS and STING, and signaling through the IFN receptor. Furthermore, SAMHD1 reduced the induction of virus-specific cytotoxic T cells in vivo. Therefore, virus restriction by SAMHD1 limits the magnitude of IFN and T cell responses. This demonstrates a competition between cell-autonomous virus control and subsequent innate and adaptive immune responses, a concept with important implications for the treatment of infection.

Goubau D, van der Veen AG, Chakravarty P, Lin R, Rogers N, Rehwinkel J, Deddouche S, Rosewell I, Hiscott J, Reis E Sousa C. 2015. Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses. Eur J Immunol, 45 (12), pp. 3386-3403. | Show Abstract | Read more

IFN-α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/β by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses.

Bridgeman A, Maelfait J, Davenne T, Partridge T, Peng Y, Mayer A, Dong T, Kaever V, Borrow P, Rehwinkel J. 2015. Viruses transfer the antiviral second messenger cGAMP between cells. Science, 349 (6253), pp. 1228-1232. | Show Abstract | Read more

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.

Cited:

25

Scopus

Rigby RE, Rehwinkel J. 2015. RNA degradation in antiviral immunity and autoimmunity Trends in Immunology, 36 (3), pp. 179-188. | Show Abstract | Read more

© 2015 The Authors. Post-transcriptional control determines the fate of cellular RNA molecules. Nonsense-mediated decay (NMD) provides quality control of mRNA, targeting faulty cellular transcripts for degradation by multiple nucleases including the RNA exosome. Recent findings have revealed a role for NMD in targeting viral RNA molecules, thereby restricting virus infection. Interestingly, NMD is also linked to immune responses at another level: mutations affecting the NMD or RNA exosome machineries cause chronic activation of defence programmes, resulting in autoimmune phenotypes. Here we place these observations in the context of other links between innate antiviral immunity and type I interferon mediated disease and examine two models: one in which expression or function of pathogen sensors is perturbed and one wherein host-derived RNA molecules with a propensity to activate such sensors accumulate.

Goubau D, Schlee M, Deddouche S, Pruijssers AJ, Zillinger T, Goldeck M, Schuberth C, Van der Veen AG, Fujimura T, Rehwinkel J et al. 2014. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates. Nature, 514 (7522), pp. 372-375. | Show Abstract | Read more

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Maelfait J, Seiradake E, Rehwinkel J. 2014. Keeping your armour intact: How HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS Prospects & Overviews J. Maelfait et al. BioEssays, 36 (7), pp. 649-657. | Show Abstract | Read more

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. © 2014 The Authors.

Rehwinkel J. 2014. Mouse knockout models for HIV-1 restriction factors. Cell Mol Life Sci, 71 (19), pp. 3749-3766. | Show Abstract | Read more

Infection of cells with human immunodeficiency virus 1 (HIV-1) is controlled by restriction factors, host proteins that counteract a variety of steps in the life cycle of this lentivirus. These include SAMHD1, APOBEC3G and tetherin, which block reverse transcription, hypermutate viral DNA and prevent progeny virus release, respectively. These and other HIV-1 restriction factors are conserved and have clear orthologues in the mouse. This review summarises studies in knockout mice lacking HIV-1 restriction factors. In vivo experiments in such animals have not only validated in vitro data obtained from cultured cells, but have also revealed new findings about the biology of these proteins. Indeed, genetic ablation of HIV-1 restriction factors in the mouse has provided evidence that restriction factors control retroviruses and other viruses in vivo and has led to new insights into the mechanisms by which these proteins counteract infection. For example, in vivo experiments in knockout mice demonstrate that virus control exerted by restriction factors can shape adaptive immune responses. Moreover, the availability of animals lacking restriction factors opens the possibility to study the function of these proteins in other contexts such as autoimmunity and cancer. Further in vivo studies of more recently identified HIV-1 restriction factors in gene targeted mice are, therefore, justified.

Rehwinkel J. 2014. Mouse knockout models for HIV-1 restriction factors. Cellular and molecular life sciences : CMLS, 71 (19), pp. 3749-3766. | Show Abstract | Read more

Infection of cells with human immunodeficiency virus 1 (HIV-1) is controlled by restriction factors, host proteins that counteract a variety of steps in the life cycle of this lentivirus. These include SAMHD1, APOBEC3G and tetherin, which block reverse transcription, hypermutate viral DNA and prevent progeny virus release, respectively. These and other HIV-1 restriction factors are conserved and have clear orthologues in the mouse. This review summarises studies in knockout mice lacking HIV-1 restriction factors. In vivo experiments in such animals have not only validated in vitro data obtained from cultured cells, but have also revealed new findings about the biology of these proteins. Indeed, genetic ablation of HIV-1 restriction factors in the mouse has provided evidence that restriction factors control retroviruses and other viruses in vivo and has led to new insights into the mechanisms by which these proteins counteract infection. For example, in vivo experiments in knockout mice demonstrate that virus control exerted by restriction factors can shape adaptive immune responses. Moreover, the availability of animals lacking restriction factors opens the possibility to study the function of these proteins in other contexts such as autoimmunity and cancer. Further in vivo studies of more recently identified HIV-1 restriction factors in gene targeted mice are, therefore, justified.

Deddouche S, Goubau D, Rehwinkel J, Chakravarty P, Begum S, Maillard PV, Borg A, Matthews N, Feng Q, van Kuppeveld FJ, Reis e Sousa C. 2014. Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells. Elife, 3 pp. e01535. | Show Abstract | Read more

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.

Clifford R, Louis T, Robbe P, Ackroyd S, Burns A, Timbs AT, Wright Colopy G, Dreau H, Sigaux F, Judde JG et al. 2014. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood, 123 (7), pp. 1021-1031. | Show Abstract | Read more

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

Cited:

63

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Rehwinkel J, Maelfait J, Bridgeman A, Rigby R, Hayward B, Liberatore RA, Bieniasz PD, Towers GJ, Moita LF, Crow YJ et al. 2013. SAMHD1-dependent retroviral control and escape in mice EMBO Journal, 32 (18), pp. 2454-2462. | Show Abstract | Read more

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1 -/- cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the K M of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism. © 2013 European Molecular Biology Organization.

Rehwinkel J. 2013. RNA sensing: the more RIG-I the merrier? EMBO Reports, 14 (9), pp. 751-752. | Read more

Rehwinkel J, Reis e Sousa C. 2013. Targeting the viral Achilles' heel: Recognition of 5'-triphosphate RNA in innate anti-viral defence Current Opinion in Microbiology, 16 (4), pp. 485-492. | Show Abstract | Read more

Some RNA virus genomes bear 5'-triphosphates, which can be recognized in the cytoplasm of infected cells by host proteins that mediate anti-viral immunity. Both the innate sensor RIG-I and the interferon-induced IFIT proteins bind to 5'-triphosphate viral RNAs. RIG-I signals for induction of interferons during RNA virus infection while IFITs sequester viral RNAs to exert an anti-viral effect. Notably, the structures of these proteins reveal both similarities and differences, which are suggestive of independent evolution towards ligand binding. 5'-triphosphates, which are absent from most RNAs in the cytosol of uninfected cells, are thus a marker of virus infection that is targeted by the innate immune system for both induction and execution of the anti-viral response. © 2013 Elsevier Ltd.

Goubau D, Rehwinkel J, Reis e Sousa C. 2010. PYHIN proteins: center stage in DNA sensing. Nat Immunol, 11 (11), pp. 984-986. | Read more

Rehwinkel J. 2010. Exposing viruses: RNA patterns sensed by RIG-I-like receptors. J Clin Immunol, 30 (4), pp. 491-495. | Show Abstract | Read more

INTRODUCTION: Virus infection triggers a multitude of innate immune pathways that limit viral replication, restrict viral spread and coordinate antigen-specific, adaptive immune responses. Type-I interferons (IFNs) play a crucial role in these events. Innate receptors expressed by the host cell recognize virus presence and signal for rapid IFN induction following infection. SCOPE: This review article discusses recent progress in our understanding of how cytoplasmic RIG-I-like receptors detect infection with RNA viruses.

Schulz O, Pichlmair A, Rehwinkel J, Rogers NC, Scheuner D, Kato H, Takeuchi O, Akira S, Kaufman RJ, Reis e Sousa C. 2010. Protein kinase R contributes to immunity against specific viruses by regulating interferon mRNA integrity. Cell Host Microbe, 7 (5), pp. 354-361. | Show Abstract | Read more

Cytosolic viral RNA recognition by the helicases RIG-I and MDA5 is considered the major pathway for IFN-alpha/beta induction in response to RNA viruses. However, other cytoplasmic RNA sensors, including the double-stranded RNA-binding protein kinase R (PKR), have been implicated in IFN-alpha/beta production, although their relative contribution and mechanism have been unclear. Using cells expressing nonfunctional PKR or reduced levels of kinase, we show that PKR is required for production of IFN-alpha/beta proteins in response to a subset of RNA viruses including encephalomyocarditis, Theiler's murine encephalomyelitis, and Semliki Forest virus, but not influenza or Sendai virus. Surprisingly, although IFN-alpha/beta mRNA induction is largely normal in PKR-deficient cells, much of that mRNA lacks the poly(A) tail, indicating that its integrity is compromised. Our results suggest that PKR plays a nonredundant role in IFN-alpha/beta production in response to some but not all viruses, in part by regulating IFN-alpha/beta mRNA stability.

Rehwinkel J, Tan CP, Goubau D, Schulz O, Pichlmair A, Bier K, Robb N, Vreede F, Barclay W, Fodor E, Reis e Sousa C. 2010. RIG-I detects viral genomic RNA during negative-strand RNA virus infection. Cell, 140 (3), pp. 397-408. | Show Abstract | Read more

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

Rehwinkel J, Reis e Sousa C. 2010. RIGorous detection: exposing virus through RNA sensing. Science, 327 (5963), pp. 284-286. | Show Abstract | Read more

Virus infection in mammals elicits a variety of defense responses that are initiated by signals from virus-sensing receptors expressed by the host. These receptors include the ubiquitously expressed RIG-I-like receptor (RLR) family of RNA helicases. RLRs are cytoplasmic proteins that act in cell-intrinsic antiviral defense by recognizing RNAs indicative of virus presence. Here, we highlight recent progress in understanding how RLRs discriminate between the RNA content of healthy versus virus-infected cells, functioning as accurate sensors of virus invasion.

Crow YJ, Rehwinkel J. 2009. Aicardi-Goutieres syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity. Hum Mol Genet, 18 (R2), pp. R130-R136. | Show Abstract | Read more

Aicardi-Goutières syndrome (AGS) is a genetically determined encephalopathy demonstrating phenotypic overlap both with the sequelae of congenital infection and with systemic lupus erythematosus (SLE). Recent molecular advances have revealed that AGS can be caused by mutations in any one of five genes, most commonly on a recessive basis but occasionally as a dominant trait. Like AGS, SLE is associated with a perturbation of type I interferon metabolism. Interestingly then, heterozygous mutations in the AGS1 gene TREX1 underlie a cutaneous subtype of SLE-called familial chilblain lupus, and mutations in TREX1 represent the single most common cause of monogenic SLE identified to date. Evidence is emerging to show that the nucleases defective in AGS are involved in removing endogenously produced nucleic acid (NA) species, and that a failure of this removal results in activation of the immune system. This hypothesis explains the phenotypic overlap of AGS with congenital infection and some aspects of SLE, where an equivalent type I interferon-mediated innate immune response is triggered by viral and self NAs, respectively. The combined efforts of clinicians, geneticists, immunologists and cell biologists are producing rapid progress in the understanding of AGS and overlapping autoimmune disorders. These studies provide important insights into the pathogenesis of SLE and beg urgent questions about the development and use of immunosuppressive therapies in AGS and related phenotypes.

Hansen KD, Lareau LF, Blanchette M, Green RE, Meng Q, Rehwinkel J, Gallusser FL, Izaurralde E, Rio DC, Dudoit S, Brenner SE. 2009. Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila. PLoS Genet, 5 (6), pp. e1000525. | Show Abstract | Read more

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA-binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD-affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD-target mRNAs had significantly longer 3' untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3' UTR length in the recognition of NMD targets in fly.

Pichlmair A, Schulz O, Tan CP, Rehwinkel J, Kato H, Takeuchi O, Akira S, Way M, Schiavo G, Reis e Sousa C. 2009. Activation of MDA5 requires higher-order RNA structures generated during virus infection. J Virol, 83 (20), pp. 10761-10769. | Show Abstract | Read more

Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-alpha/beta) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-alpha/beta production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-alpha/beta production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.

Eulalio A, Huntzinger E, Nishihara T, Rehwinkel J, Fauser M, Izaurralde E. 2009. Deadenylation is a widespread effect of miRNA regulation. RNA, 15 (1), pp. 21-32. | Show Abstract | Read more

miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. In animal cells, degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1, or NOT1. We show that approximately 60% of AGO1 targets are regulated by CAF1 and/or NOT1, indicating that deadenylation is a widespread effect of miRNA regulation. However, neither a poly(A) tail nor mRNA circularization are required for silencing, because mRNAs whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo. We show further that miRNAs trigger mRNA degradation, even when binding by 40S ribosomal subunits is inhibited in cis. These results indicate that miRNAs promote mRNA decay by altering mRNP composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.

Eulalio A, Rehwinkel J, Stricker M, Huntzinger E, Yang SF, Doerks T, Dorner S, Bork P, Boutros M, Izaurralde E. 2007. Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing. Genes Dev, 21 (20), pp. 2558-2570. | Show Abstract | Read more

microRNAs (miRNAs) silence gene expression by suppressing protein production and/or by promoting mRNA decay. To elucidate how silencing is accomplished, we screened an RNA interference library for suppressors of miRNA-mediated regulation in Drosophila melanogaster cells. In addition to proteins known to be required for miRNA biogenesis and function (i.e., Drosha, Pasha, Dicer-1, AGO1, and GW182), the screen identified the decapping activator Ge-1 as being required for silencing by miRNAs. Depleting Ge-1 alone and/or in combination with other decapping activators (e.g., DCP1, EDC3, HPat, or Me31B) suppresses silencing of several miRNA targets, indicating that miRNAs elicit mRNA decapping. A comparison of gene expression profiles in cells depleted of AGO1 or of individual decapping activators shows that approximately 15% of AGO1-targets are also regulated by Ge-1, DCP1, and HPat, whereas 5% are dependent on EDC3 and LSm1-7. These percentages are underestimated because decapping activators are partially redundant. Furthermore, in the absence of active translation, some miRNA targets are stabilized, whereas others continue to be degraded in a miRNA-dependent manner. These findings suggest that miRNAs mediate post-transcriptional gene silencing by more than one mechanism.

Behm-Ansmant I, Kashima I, Rehwinkel J, Saulière J, Wittkopp N, Izaurralde E. 2007. mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett, 581 (15), pp. 2845-2853. | Show Abstract | Read more

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway which ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), thereby preventing the accumulation of truncated and potentially harmful proteins. In this way, the NMD pathway contributes to suppressing or exacerbating the clinical manifestations of specific human genetic disorders. Studies in model organisms have led to the identification of the effectors of the NMD pathway, and illuminated the mechanisms by which premature stops are discriminated from natural stops, so that only the former trigger rapid mRNA degradation. These studies are providing important insights that will aid the development of new treatments for at least some human genetic diseases.

Behm-Ansmant I, Gatfield D, Rehwinkel J, Hilgers V, Izaurralde E. 2007. A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay. EMBO J, 26 (6), pp. 1591-1601. | Show Abstract | Read more

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs with premature translation termination codons (PTCs). The mechanisms by which PTCs and natural stop codons are discriminated remain unclear. We show that the position of stops relative to the poly(A) tail (and thus of PABPC1) is a critical determinant for PTC definition in Drosophila melanogaster. Indeed, tethering of PABPC1 downstream of a PTC abolishes NMD. Conversely, natural stops trigger NMD when the length of the 3' UTR is increased. However, many endogenous transcripts with exceptionally long 3' UTRs escape NMD, suggesting that the increase in 3' UTR length has co-evolved with the acquisition of features that suppress NMD. We provide evidence for the existence of 3' UTRs conferring immunity to NMD. We also show that PABPC1 binding is sufficient for PTC recognition, regardless of cleavage or polyadenylation. The role of PABPC1 in NMD must go beyond that of providing positional information for PTC definition, because its depletion suppresses NMD under conditions in which translation efficiency is not affected. These findings reveal a conserved role for PABPC1 in mRNA surveillance.

Rehwinkel J, Raes J, Izaurralde E. 2006. Nonsense-mediated mRNA decay: Target genes and functional diversification of effectors. Trends Biochem Sci, 31 (11), pp. 639-646. | Show Abstract | Read more

Recent genome-wide identification of nonsense-mediated mRNA decay (NMD) targets in yeast, fruitfly and human cells has provided insight into the biological functions and evolution of this mRNA quality control mechanism, revealing that NMD post-transcriptionally regulates an important fraction of the transcriptome. NMD targets are associated with a broad range of biological processes, but most of these targets are not encoded by orthologous genes across different species. Yeast and fruitfly NMD effectors regulate common targets in concert, but parallel pathways have evolved in humans, whereby NMD effectors have acquired additional functions. Thus, the phenotypic differences observed across species after inhibition of NMD are driven not only by the functional diversification of NMD effectors but also by changes in the repertoire of regulated genes.

Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E. 2006. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev, 20 (14), pp. 1885-1898. | Show Abstract | Read more

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. We further show that the N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. Our findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets.

Rehwinkel J, Natalin P, Stark A, Brennecke J, Cohen SM, Izaurralde E. 2006. Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster. Mol Cell Biol, 26 (8), pp. 2965-2975. | Show Abstract | Read more

RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, we examined mRNA expression profiles in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. We also profiled cells depleted of the miRNA-processing enzyme Drosha. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought.

Behm-Ansmant I, Rehwinkel J, Izaurralde E. 2006. MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb Symp Quant Biol, 71 pp. 523-530. | Show Abstract | Read more

microRNAs (miRNAs) represent a novel class of genome-encoded eukaryotic regulatory RNAs that silence gene expression posttranscriptionally. Although the proteins mediating miRNA biogenesis and function have been identified, the precise mechanism by which miRNAs regulate the expression of target mRNAs remains unclear. We summarize recent work from our laboratory demonstrating that miRNAs silence gene expression by at least two independent mechanisms: by repressing translation and/or by promoting mRNA degradation. In Drosophila, both mechanisms require Argonaute 1 (AGO1) and the P-body component GW182. Moreover, mRNA degradation by miRNAs is effected by the enzymes involved in general mRNA decay, including deadenylases and decapping enzymes, which also localize to P bodies. Our findings suggest a model for miRNA function in which AGO1 associates with miRNA targets through miRNA:mRNA base-pairing interactions. GW182 interacts with AGO1 and recruits deadenylases and decapping enzymes, leading to mRNA degradation. However, not all miRNA targets are degraded: Some stay in a translationally silent state, from which they may eventually be released. We propose that the final outcome of miRNA regulation (i.e., degradation vs. translational repression) is influenced by other RNA-binding proteins interacting with the targeted mRNA.

Rehwinkel J, Behm-Ansmant I, Gatfield D, Izaurralde E. 2005. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA, 11 (11), pp. 1640-1647. | Show Abstract | Read more

In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins that are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional processes we depleted P-body or essential pathway components from Drosophila cells and analyzed the effects of these depletions on the expression of reporter constructs, allowing us to monitor specifically NMD, RNAi, or miRNA function. We show that the RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. Our analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila.

Rehwinkel J, Letunic I, Raes J, Bork P, Izaurralde E. 2005. Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets. RNA, 11 (10), pp. 1530-1544. | Show Abstract | Read more

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades mRNAs containing nonsense codons, and regulates the expression of naturally occurring transcripts. While NMD is not essential in yeast or nematodes, UPF1, a key NMD effector, is essential in mice. Here we show that NMD components are required for cell proliferation in Drosophila. This raises the question of whether NMD effectors diverged functionally during evolution. To address this question, we examined expression profiles in Drosophila cells depleted of all known metazoan NMD components. We show that UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6 regulate in concert the expression of a cohort of genes with functions in a wide range of cellular activities, including cell cycle progression. Only a few transcripts were regulated exclusively by individual factors, suggesting that these proteins act mainly in the NMD pathway and their role in mRNA decay has not diverged substantially. Finally, the vast majority of NMD targets in Drosophila are not orthologs of targets previously identified in yeast or human cells. Thus phenotypic differences observed across species following inhibition of NMD can be largely attributed to changes in the repertoire of regulated genes.

Jínek M, Rehwinkel J, Lazarus BD, Izaurralde E, Hanover JA, Conti E. 2004. The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha. Nat Struct Mol Biol, 11 (10), pp. 1001-1007. | Show Abstract | Read more

Addition of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin alpha. Based on this structural similarity, we propose that OGT uses an analogous molecular mechanism to recognize its targets.

Rehwinkel J, Herold A, Gari K, Köcher T, Rode M, Ciccarelli FL, Wilm M, Izaurralde E. 2004. Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster. Nat Struct Mol Biol, 11 (6), pp. 558-566. | Show Abstract | Read more

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

Erdal E, Haider S, Rehwinkel J, Harris AL, McHugh PJ. 2017. A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1. Genes Dev, 31 (4), pp. 353-369. | Show Abstract | Read more

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.

Maelfait J, Bridgeman A, Benlahrech A, Cursi C, Rehwinkel J. 2016. Restriction by SAMHD1 Limits cGAS/STING-Dependent Innate and Adaptive Immune Responses to HIV-1. Cell Rep, 16 (6), pp. 1492-1501. | Show Abstract | Read more

SAMHD1 is a restriction factor for HIV-1 infection. SAMHD1 mutations cause the autoinflammatory Aicardi-Goutières syndrome that is characterized by chronic type I interferon (IFN) secretion. We show that the spontaneous IFN response in SAMHD1-deficient cells and mice requires the cGAS/STING cytosolic DNA-sensing pathway. We provide genetic evidence that cell-autonomous control of lentivirus infection in myeloid cells by SAMHD1 limits virus-induced production of IFNs and the induction of co-stimulatory markers. This program of myeloid cell activation required reverse transcription, cGAS and STING, and signaling through the IFN receptor. Furthermore, SAMHD1 reduced the induction of virus-specific cytotoxic T cells in vivo. Therefore, virus restriction by SAMHD1 limits the magnitude of IFN and T cell responses. This demonstrates a competition between cell-autonomous virus control and subsequent innate and adaptive immune responses, a concept with important implications for the treatment of infection.

Bridgeman A, Maelfait J, Davenne T, Partridge T, Peng Y, Mayer A, Dong T, Kaever V, Borrow P, Rehwinkel J. 2015. Viruses transfer the antiviral second messenger cGAMP between cells. Science, 349 (6253), pp. 1228-1232. | Show Abstract | Read more

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.

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Rigby RE, Rehwinkel J. 2015. RNA degradation in antiviral immunity and autoimmunity Trends in Immunology, 36 (3), pp. 179-188. | Show Abstract | Read more

© 2015 The Authors. Post-transcriptional control determines the fate of cellular RNA molecules. Nonsense-mediated decay (NMD) provides quality control of mRNA, targeting faulty cellular transcripts for degradation by multiple nucleases including the RNA exosome. Recent findings have revealed a role for NMD in targeting viral RNA molecules, thereby restricting virus infection. Interestingly, NMD is also linked to immune responses at another level: mutations affecting the NMD or RNA exosome machineries cause chronic activation of defence programmes, resulting in autoimmune phenotypes. Here we place these observations in the context of other links between innate antiviral immunity and type I interferon mediated disease and examine two models: one in which expression or function of pathogen sensors is perturbed and one wherein host-derived RNA molecules with a propensity to activate such sensors accumulate.

Goubau D, Schlee M, Deddouche S, Pruijssers AJ, Zillinger T, Goldeck M, Schuberth C, Van der Veen AG, Fujimura T, Rehwinkel J et al. 2014. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates. Nature, 514 (7522), pp. 372-375. | Show Abstract | Read more

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Maelfait J, Seiradake E, Rehwinkel J. 2014. Keeping your armour intact: How HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS Prospects &amp; Overviews J. Maelfait et al. BioEssays, 36 (7), pp. 649-657. | Show Abstract | Read more

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. © 2014 The Authors.

Rehwinkel J. 2014. Mouse knockout models for HIV-1 restriction factors. Cell Mol Life Sci, 71 (19), pp. 3749-3766. | Show Abstract | Read more

Infection of cells with human immunodeficiency virus 1 (HIV-1) is controlled by restriction factors, host proteins that counteract a variety of steps in the life cycle of this lentivirus. These include SAMHD1, APOBEC3G and tetherin, which block reverse transcription, hypermutate viral DNA and prevent progeny virus release, respectively. These and other HIV-1 restriction factors are conserved and have clear orthologues in the mouse. This review summarises studies in knockout mice lacking HIV-1 restriction factors. In vivo experiments in such animals have not only validated in vitro data obtained from cultured cells, but have also revealed new findings about the biology of these proteins. Indeed, genetic ablation of HIV-1 restriction factors in the mouse has provided evidence that restriction factors control retroviruses and other viruses in vivo and has led to new insights into the mechanisms by which these proteins counteract infection. For example, in vivo experiments in knockout mice demonstrate that virus control exerted by restriction factors can shape adaptive immune responses. Moreover, the availability of animals lacking restriction factors opens the possibility to study the function of these proteins in other contexts such as autoimmunity and cancer. Further in vivo studies of more recently identified HIV-1 restriction factors in gene targeted mice are, therefore, justified.

Rehwinkel J. 2014. Mouse knockout models for HIV-1 restriction factors. Cellular and molecular life sciences : CMLS, 71 (19), pp. 3749-3766. | Show Abstract | Read more

Infection of cells with human immunodeficiency virus 1 (HIV-1) is controlled by restriction factors, host proteins that counteract a variety of steps in the life cycle of this lentivirus. These include SAMHD1, APOBEC3G and tetherin, which block reverse transcription, hypermutate viral DNA and prevent progeny virus release, respectively. These and other HIV-1 restriction factors are conserved and have clear orthologues in the mouse. This review summarises studies in knockout mice lacking HIV-1 restriction factors. In vivo experiments in such animals have not only validated in vitro data obtained from cultured cells, but have also revealed new findings about the biology of these proteins. Indeed, genetic ablation of HIV-1 restriction factors in the mouse has provided evidence that restriction factors control retroviruses and other viruses in vivo and has led to new insights into the mechanisms by which these proteins counteract infection. For example, in vivo experiments in knockout mice demonstrate that virus control exerted by restriction factors can shape adaptive immune responses. Moreover, the availability of animals lacking restriction factors opens the possibility to study the function of these proteins in other contexts such as autoimmunity and cancer. Further in vivo studies of more recently identified HIV-1 restriction factors in gene targeted mice are, therefore, justified.

Deddouche S, Goubau D, Rehwinkel J, Chakravarty P, Begum S, Maillard PV, Borg A, Matthews N, Feng Q, van Kuppeveld FJ, Reis e Sousa C. 2014. Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells. Elife, 3 pp. e01535. | Show Abstract | Read more

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.

Clifford R, Louis T, Robbe P, Ackroyd S, Burns A, Timbs AT, Wright Colopy G, Dreau H, Sigaux F, Judde JG et al. 2014. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood, 123 (7), pp. 1021-1031. | Show Abstract | Read more

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

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Scopus

Rehwinkel J, Maelfait J, Bridgeman A, Rigby R, Hayward B, Liberatore RA, Bieniasz PD, Towers GJ, Moita LF, Crow YJ et al. 2013. SAMHD1-dependent retroviral control and escape in mice EMBO Journal, 32 (18), pp. 2454-2462. | Show Abstract | Read more

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1 -/- cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the K M of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism. © 2013 European Molecular Biology Organization.

Rehwinkel J. 2013. RNA sensing: the more RIG-I the merrier? EMBO Reports, 14 (9), pp. 751-752. | Read more

Rehwinkel J, Reis e Sousa C. 2013. Targeting the viral Achilles' heel: Recognition of 5'-triphosphate RNA in innate anti-viral defence Current Opinion in Microbiology, 16 (4), pp. 485-492. | Show Abstract | Read more

Some RNA virus genomes bear 5'-triphosphates, which can be recognized in the cytoplasm of infected cells by host proteins that mediate anti-viral immunity. Both the innate sensor RIG-I and the interferon-induced IFIT proteins bind to 5'-triphosphate viral RNAs. RIG-I signals for induction of interferons during RNA virus infection while IFITs sequester viral RNAs to exert an anti-viral effect. Notably, the structures of these proteins reveal both similarities and differences, which are suggestive of independent evolution towards ligand binding. 5'-triphosphates, which are absent from most RNAs in the cytosol of uninfected cells, are thus a marker of virus infection that is targeted by the innate immune system for both induction and execution of the anti-viral response. © 2013 Elsevier Ltd.

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