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The molecular basis of ligand recognition by RIG-I and viral escape strategies

English title The molecular basis of ligand recognition by RIG-I and viral escape strategies
Applicant Garcin Dominique
Number 135467
Funding scheme Project funding (Div. I-III)
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Molecular Biology
Start/End 01.04.2011 - 31.03.2015
Approved amount 415'032.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (6)

Innate immunity; PAMP ; PRR; Paramyxovirus; Arenavirus; IFN antagonists

Lay Summary (English)

Lead
Lay summary

The result is a delicate balance, the consequence ofan endless co-evolution. When this fragile balance was broken in the past,during epidemics, the consequences were often dramatic. The intimate knowledgeof the interactions between virus and its host will hopefully maintain thisbalance on our side. There is clearlysomething fascinating in the eternal battle between the virus and its host. Viruses, the most abundant pathogens, are obligatoryparasites that largely depend on the cellular machinery for their replication,so there are few specific viral targets for antiviral therapies. Besides,escape mutants resistant to antiviral agents generally emerge rapidly for RNAviruses, due to their high mutation rate. In response to viralinfection, organisms have evolved efficient and sophisticated host-defensemechanisms to sense viruses and block their replication and spread. Innateimmunity represents one of the first lines of defense against viral infections,preventing their propagation, and preparing the inflammatory and the adaptiveimmune response. Detailed knowledge of how this first line of defence is established, andhow viruses escape these antiviral defences, is critical to understanding howto prevent viral infection. Innate immunity starts with the specific detectionof a pathogen, and in the case of viruses whose structural components arederived entirely from the host-cell, this detection represent a real challenge.The basis of all immune reaction is the discrimination between self and non-self,based on the detection of pathogen-associated molecular pattern (PAMPs). In thecase of viruses, the main determinant of this specific detection is viralnucleic-acids. This detection is done by specific pattern recognition receptors(PRRs), expressed not only in immune cells but also in all cells susceptible tobe infected. RNA virus infections are sensed primarily by two DExD/H boxhelicases, RIG-I and MDA-5 in response to two RNA PAMPs, namely dsRNA (e.g.,poly-I:poly-C, or poly-I/C) and 5’ ppp-RNA. These RNAs can act as PAMPs becausetheir cytoplasmic presence is thought to be restricted to virus infection.RIG-I senses most RNA virus infections including important pathogens such asInfluenza viruses, paramyxoviruses and Arenaviruses. Our scientific project isfocused on the nature of these PAMPs in viral infections and how viruses canescape this detection and is articulated around two axes :

i)Molecular basis ofligand recognition by RIG-I and escape viral strategies.involved in blocking RIG-I activation and whose function clearly dependson the integrity of their dsRNA binding domain. In vitro and invivo RNA binding experiments to RIG-I will be performed using either mutated bacteriallypurified RIG-I or transfected RIG-I. Arenavirus infection will be used tofurther characterize the ligand sensed by RIG-I and to study the Arenavirus decoystrategy in the context of a viral infection. In vivo and in vitro RNA bindingassays will also be used to characterize the RNA ligand of viral proteins (suchas vaccinia virus E3L, influenza virus NS1…)

ii)The “real” PAMP inparamyxovirus infection. The exact nature of the PAMP(s) generated duringParamyxovirus infection is still a subject of debate. Virus stocks of eithernon defective genomes or internal deletion defective interfering genomes (DI)or copy back DI genomes carrying mutations in the L terminal sequence will begenerated and analyzed for their ability to induce IFN-ß. The nature ofthe PAMP bound to RIG-I will be further characterized in those infections.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Name Institute

Publications

Publication
The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing.
Pourcelot Marie, Zemirli Naima, Silva Da Costa Leandro, Loyant Roxane, Garcin Dominique, Vitour Damien, Munitic Ivana, Vazquez Aimé, Arnoult Damien (2016), The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing., in BMC biology, 14, 69-69.
Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation.
Anchisi Stéphanie, Guerra Jessica, Mottet-Osman Geneviève, Garcin Dominique (2015), Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation., in Journal of virology, 90(1), 586-90.
RIG-I ATPase activity and discrimination of self-RNA versus non-self-RNA.
Anchisi Stéphanie, Guerra Jessica, Garcin Dominique (2015), RIG-I ATPase activity and discrimination of self-RNA versus non-self-RNA., in mBio, 6(2), 02349-02349.
TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State.
Széles Lajos, Meissner Felix, Dunand-Sauthier Isabelle, Thelemann Christoph, Hersch Micha, Singovski Simon, Haller Sergio, Gobet Florian, Fuertes Marraco Silvia A, Mann Matthias, Garcin Dominique, Acha-Orbea Hans, Reith Walter (2015), TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State., in Journal of immunology (Baltimore, Md. : 1950), J Immunol. 2015 Jun 22. pii: 1402033. [Epub ahead (J Immunol.), J Immunol.-J Immunol..
γHV68 vGAT: a viral pseudoenzyme pimping for PAMPs.
Kolakofsky Daniel, Garcin Dominique (2015), γHV68 vGAT: a viral pseudoenzyme pimping for PAMPs., in Molecular cell, 58(1), 3-4.
MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors.
Castanier Céline, Zemirli Naima, Portier Alain, Garcin Dominique, Bidère Nicolas, Vazquez Aimé, Arnoult Damien (2012), MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors., in BMC biology, 10, 44-44.
Human parainfluenza virus type 2 L protein regions required for interaction with other viral proteins and mRNA capping.
Nishio Machiko, Tsurudome Masato, Garcin Dominique, Komada Hiroshi, Ito Morihiro, Le Mercier Philippe, Nosaka Tetsuya, Kolakofsky Daniel (2011), Human parainfluenza virus type 2 L protein regions required for interaction with other viral proteins and mRNA capping., in Journal of virology, 85(2), 725-32.
Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus.
Le Thuc-vy L, Mironova Elena, Garcin Dominique, Compans Richard W (2011), Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus., in PloS one, 6(4), 18780-18780.
Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus.
Rawling Joanna, Cano Olga, Garcin Dominique, Kolakofsky Daniel, Melero José A (2011), Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus., in Journal of virology, 85(6), 2771-80.
Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys.
Marq Jean-Baptiste, Hausmann Stéphane, Veillard Nicolas, Kolakofsky Daniel, Garcin Dominique (2011), Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys., in The Journal of biological chemistry, 286(8), 6108-16.

Scientific events



Self-organised

Title Date Place
16th Negative strand virus meeting 14.06.2015 Sienna, Italy
15th Negative Strand Virus meeting 16.06.2013 Grenada, Spain

Associated projects

Number Title Start Funding scheme
116114 Interactions of Paramyxoviruses and host cells 01.04.2007 Project funding (Div. I-III)
163129 RIG-I sensing of viral infections: beyond discrimination between self and non-self RNAs. 01.10.2015 Project funding (Div. I-III)

Abstract

The molecular basis of ligand recognition by RIG-I and viral escape strategies1.Summary1.1BackgroundViral infections are responsible for extensive human and animal suffering and death, resulting in heavy human and economic cost. Very few antiviral molecules are available and more importantly escape mutants resistant to existing antiviral agents generally emerge rapidly for RNA viruses, due to their high mutation rate. On the other hand, one of the first line of defense against viral infection is innate immunity. Therefore detailed knowledge of how this first line of defense is established and how viruses escape these antiviral defenses, is critical to understand how to prevent viral infection. Recent developments in this field now hold the promise of fighting viral infections by enhancing the innate immune response. Innate immunity starts with the specific detection of a pathogen. Detection of specific “non-self” viral nucleic acid in the cytoplasm involves cytosolic pattern recognition receptors (PRRs) including the RNA-sensing RIG-like helicases (RLHs), RIG-I, MDA-5, LGP2.1.2Working hypothesesViral strategies to escape detection by RIG-I clearly represent an interesting approach. Viruses are probably the best molecular biologists; they are professionals in handling cellular metabolic pathways, either to escape or fight these pathways, or to divert them to their own benefit. They are thus an invaluable source of information on these cellular pathways. Work from our laboratory and others has led us to elaborate a working model in which RIG-I first binds dsRNA, then moves in an ATP dependent fashion to locate and interact with its end. A blunt 5’ppp-dsRNA-end leads to productive RIG-I conformational changes and full IFN-ß induction(at least for short dsRNAs). For long dsRNAs devoid of blunt 5’ppp-ends, the stabilization of the RIG-I/dsRNA (required for activation of RIG-I and IFN-ß induction), appears to be somehow related to the length of this dsRNA. We propose to test this model and also to study what exactly are the pathogen associated molecular patterns (PAMPs) of paramyxovirus and arenavirus infections.1.3Specific aims and experimental designWe will develop two important aspects of our work: i)Molecular basis of ligand recognition by RIG-I and escape viral strategies. In vitro and in vivo RNA binding experiments to RIG-I will be performed using either mutated bacterially purified RIG-I or transfected RIG-I. Arenavirus infection will be used to further characterize the ligand sensed by RIG-I and to study the Arenavirus decoy strategy in the context of a viral infection. In vivo and in vitro RNA binding assays will also be used to characterize the RNA ligand of viral proteins (such as vaccinia virus E3L, influenza virus NS1…) involved in blocking RIG-I activation and whose function clearly depends on the integrity of their dsRNA binding domain. ii)The “real” PAMP in paramyxovirus infection. The exact nature of the PAMP(s) generated during Paramyxovirus infection is still a subject of debate. Virus stocks of either non defective genomes or internal deletion defective interfering genomes (DI) or copy back DI genomes carrying mutations in the L terminal sequence will be generated and analyzed for their ability to induce IFN-ß. The nature of the PAMP bound to RIG-I will be further characterized in those infections.
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