Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)-competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo-electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability

Rôle des méthylations des ARN viraux dans la réponse antivirale

Ces dernières années, des changements épitranscriptomiques ont été détectés dans de nombreux ARN viraux, mais la fonction biologique de la plupart reste mal comprise. Parmi ces modifications, les méthylations d'ARN sont des marques clés. La première partie de ce manuscrit traite de la 2'O-méthylation de l'ARN, qui est un marqueur du "soi" permettant de discriminer les ARNm des pathogènes. Nous avons d'abord démontré que la 2'O-méthylation des ARN altère la dégradation médiée par ISG20. L'analyse structure-fonction indique que cette inhibition dérive d'un mécanisme d'encombrement stérique. De plus, les génomes hypo-méthylés du VIH-1 produits dans les cellules FTSJ3-KO sont plus susceptibles d'être dégradés par ISG20 que ceux des cellules exprimant FTSJ3. Par conséquent, la transcription inverse et la production de virus hypométhylés sont altérées, démontrant l'effet antagoniste direct de la 2'O-méthylation sur l'activité antivirale médiée par ISG20. Dans la deuxième partie du manuscrit, nous avons caractérisé la protéine non structurale CoV 14 (nsp14), une protéine bifonctionnelle avec un domaine ExoN N-terminal 3′ à 5′ et un domaine N7-MTase C-terminal impliqué dans le coiffage de l'ARNm viral. Nous avons identifié plusieurs résidus impliqués dans la formation de la poche catalytique N7-MTase et évalué leur importance pour l'activité enzymatique in vitro et la réplication virale. Nos résultats soulignent la N7-MTase en tant qu'enzyme importante pour la réplication des bétacoronavirus et définissent les résidus clés de sa poche catalytique qui peuvent être ciblés pour concevoir des inhibiteurs avec un spectre d'activité pan-coronavirus potentiel.In recent years, epitranscriptomic modifications have been detected in numerous viral RNA, but the physiological function of most of them remains barely understood. Among these modifications, RNA methylations are an important modification induced by specific viral or cellular RNA methyltransferases. The first part of this manuscript focuses on RNA 2’O-methylation, which is a "self" marker that allows discriminating between host cell and pathogen mRNAs. Here, we showed by an In vitro assay that ISG20-mediated decay of 2'O-methylated RNA is impaired. Structure-function analysis indicated that this inhibition results from a steric hindrance mechanism. Moreover, hypo-methylated HIV-1 genomes produced in FTSJ3-KO cells were more prone to degradation by ISG20 than those produced in cells that express wild-type FTSJ3. Consequently, the retrotranscription and production of hypomethylated viruses were impaired, demonstrating the direct antagonist effect of 2’O-methylation on ISG20-mediated antiviral activity. In the second part of the manuscript, we characterized the CoV nonstructural protein 14 (nsp14) which is a bifunctional protein harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal N7-MTase domain, which is presumably involved in viral mRNA capping. We identified several residues involved in the formation of the N7-MTase catalytic pocket and assessed their importance for in vitro enzymatic activity and virus replication. Our results highlighted the N7-MTase as a critical enzyme for betacoronavirus replication and defined key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum

Interplay of RNA 2′-O-methylations with viral replication

International audienceHighlights • Viral RNA genomes are modified by epitranscriptomic marks, such as 2′-Omethylation. • 2′-O-methylations are self-markers hijacked by viruses to avoid immune sensing. • Modified viral RNA counteract restriction by interferon-stimulated genes. • New insights suggest that 2′-O-methylations also impair viral replication

Protéine 2C d’Entérovirus : protéine clé de la réplication virale et cible antivirale

Enteroviruses (EVs) include many human pathogens of increasing public health concern. These EVs are often associated with mild clinical manifestations, but they can lead to serious complications such as encephalitis, meningitis, pneumonia, myocarditis or poliomyelitis. Despite significant advances, there is no approved antiviral therapy for the treatment of enterovirus infections. Due to the high genotypic diversity of EVs, molecules targeting highly conserved viral proteins may be considered for developing a pan-EV treatment. In this regard,the ATPase/Helicase 2C, which is a highly conserved non-structural protein among EVs, has essential functions for viral replication and is therefore an attractive antiviral target. Recent functional and structural studies on the 2C protein led to the identification of molecules showing ex vivo anti-EV activity and associated with resistance mutations on the coding sequence of the 2C protein. This review presents the current state of knowledge about the 2C protein from an antiviral target perspective and the mode of action of specific inhibitors for this therapeutic target.Les entérovirus (EV) comprennent de nombreux agents pathogènes humains de plus en plus préoccupants en matière de santé publique. Ces EV sont souvent associés à des manifestations cliniques bénignes, mais peuvent entraîner des complications graves telles que des encéphalites, des méningites, des pneumonies, des myocardites et des poliomyélites. Malgré d’importantes avancées, il n’existe pas de thérapie antivirale approuvée pour le traitement des infections à entérovirus. De par la grande diversité génotypique des EV, des molécules ciblant des protéines virales fortement conservées peuvent être envisagées pour le développement d’antiviraux pan-EV. À cet égard, l’ATPase/hélicase 2C, qui est une protéine non structurale hautement conservée chez les EVs, possède des fonctions essentielles à la réplication virale, et s’avère donc une cible antivirale attractive. De récentes études fonctionnelles et structurales sur la protéine 2C ont permis l’identification de molécules démontrant une activité anti-EV ex vivo et associées à des mutations de résistance sur la séquence codante de la protéine 2C. Cette revue présente un état des lieux des connaissances actuelles sur la protéine 2C dans une perspective de cible antivirale et sur le mode d’action d’inhibiteurs spécifiques de cette cible thérapeutique

Protéine 2C d’Entérovirus : protéine clé de la réplication virale et cible antivirale

International audienceEnteroviruses (EVs) include many human pathogens of increasing public health concern. These EVs are often associated with mild clinical manifestations, but they can lead to serious complications such as encephalitis, meningitis, pneumonia, myocarditis or poliomyelitis. Despite significant advances, there is no approved antiviral therapy for the treatment of enterovirus infections. Due to the high genotypic diversity of EVs, molecules targeting highly conserved viral proteins may be considered for developing a pan-EV treatment. In this regard, the ATPase/Helicase 2C, which is a highly conserved non-structural protein among EVs, has essential functions for viral replication and is therefore an attractive antiviral target. Recent functional and structural studies on the 2C protein led to the identification of molecules showing ex vivo anti-EV activity and associated with resistance mutations on the coding sequence of the 2C protein. This review presents the current state of knowledge about the 2C protein from an antiviral target perspective and the mode of action of specific inhibitors for this therapeutic target.Les entérovirus (EV) comprennent de nombreux agents pathogènes humains de plus en plus préoccupants en matière de santé publique. Ces EV sont souvent associés à des manifestations cliniques bénignes, mais peuvent entraîner des complications graves telles que des encéphalites, des méningites, des pneumonies, des myocardites et des poliomyélites. Malgré d’importantes avancées, il n'existe pas de thérapie antivirale approuvée pour le traitement des infections à entérovirus. De par la grande diversité génotypique des EV, des molécules ciblant des protéines virales fortement conservées peuvent être envisagées pour le développement d’antiviraux pan-EV. À cet égard, l’ATPase/Hélicase 2C, qui est une protéine non structurale hautement conservée chez les EVs, possède des fonctions essentielles à la réplication virale, et s’avère donc une cible antivirale attractive. De récentes études fonctionnelles et structurales sur la protéine 2C ont permis l’identification de molécules démontrant une activité anti-EV ex vivo et associées à des mutations de résistance sur la séquence codante de la protéine 2C. Cette revue présente un état des lieux des connaissances actuelles sur la protéine 2C dans une perspective de cible antivirale et sur le mode d'action d'inhibiteurs spécifiques de cette cible thérapeutique

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication

Abstract As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5’ exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3′-to-5′ exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan -coronaviral activity spectrum. Significance Statement The ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2

Internal RNA 2′O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect

International audienceRNA 2′O-methylation is a ‘self’ epitranscriptomic modification allowing discrimination between host and pathogen. Indeed, human immunodeficiency virus 1 (HIV-1) induces 2′O-methylation of its genome by recruiting the cellular FTSJ3 methyltransferase, thereby impairing detection by RIG-like receptors. Here, we show that RNA 2′O-methylations interfere with the antiviral activity of interferon-stimulated gene 20-kDa protein (ISG20). Biochemical experiments showed that ISG20-mediated degradation of 2′O-methylated RNA pauses two nucleotides upstream of and at the methylated residue. Structure-function analysis indicated that this inhibition is due to steric clash between ISG20 R53 and D90 residues and the 2′O-methylated nucleotide. We confirmed that hypomethylated HIV-1 genomes produced in FTSJ3-KO cells were more prone to in vitro degradation by ISG20 than those produced in cells expressing FTSJ3. Finally, we found that reverse-transcription of hypomethylated HIV-1 was impaired in T cells by interferon-induced ISG20, demonstrating the direct antagonist effect of 2′O-methylation on ISG20-mediated antiviral activity

A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases

Abstract Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses. Highlights Arenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary origin Arenaviridae and Coronaviridae exoribonuclease is an attractive target for drug development We present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarization We validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleases We determined the IC 50 by fluorescence polarization of one inhibitor common to the three viruses