Implication des protéines non structurales des alphavirus dans l'évasion virale : applications comme nouvelles cibles pour des agents antiviraux

Au cours de l'infection virale, la présence d'intermédiaires de réplication de l'ARN viral dans le cytoplasme déclenche la réponse antivirale. l'ARN viral est reconnu par des récepteurs cytoplasmiques tels que: (i) PKR, qui médie la signalisation pour la production d' IFN. (ii) OAS, conduisant à la synthèse d'oligonucléotides courts 2-5 (2-5A) pour activer la RNase L. (iii) les senseurs RIG-I et MDA5, favorisant l'induction des ISG. Ici, nous étudions le rôle des nsps dans l'évasion virale. Chez les alphavirus, nous avons étudié l'enzyme de coiffage nsp1, portant des activités méthyl- et guanylyltransférase. Nous avons caractérisé le rôle des résidus impliqués dans la résistance à la ribavirine et mis en lumière les spécificités du substrat d'ARN pour la réaction de la guanylyltransférase. Pour le virus de l'hépatite E, nos données montrent que les domaines contenant une caractéristique de l'activité phosphodiestérase, peuvent modifier/dégrader 2-5A. De plus, ces domaines inhibent la production d' IFN dans les cellules de mammifères. Ces résultats suggèrent que le virus pourrait échapper à la réponse immunitaire innée par l'inhibition du système OAS/RNAse L. Nous avons aussi caractérisé le Macro domaine de SARS-CoV-2. Les Macro domaines peuvent se lier et hydrolyser l’ADP-ribose. Grâce à une étude de mutagenèse, nous avons mis en évidence l'importance de l'orientation de l'ADP-ribose dans la clé de liaison pour l'activité. De plus, nous avons conçu et testé des inhibiteurs potentiels, identifiant les déterminants moléculaires clés pour les études d'optimisation de ces inhibiteurs. Finalement, ces études peuvent conduire plus largement au développement d'outils antiviraux.In the course of the viral infection, the presence of viral RNA replication intermediates in the cytoplasm triggers the antiviral response. Hence, viral RNA is recognized by cytoplasmic receptors such as: (i) PKR, which inhibits protein translation and mediates signaling to enhance IFN production. (ii) OAS, leading to the synthesis of short 2-5 oligonucleotides (2-5A) to activate RNase L. (iii) RIG-I and MDA5 sensors, promoting the induction of ISGs. Here we investigate the role of nsps from alphavirus-like super family and Coronavirus in viral escape. In alphavirus, we studied the capping enzyme nsp1, carrying methyl-and guanylyltransferase activities. We characterized the role of residues involved in ribavirin resistance and broth some light on the RNA substrate requirements for guanylyltransferase reaction. For Hepatitis E virus, our data show that the domains containing a hallmark of phosphodiesterase activity, can modify/degrade 2-5A. Also, these domains inhibite IFN production in mammalian cells. These findings suggest that HEV could escape innate immune response through the inhibition of OAS/RNAse L system. In parallel, we characterized SARS-CoV-2 Macro domain. Macro domains can bind and hydrolyze ADP-ribose derivatives, important for IFN production. Through mutagenesis study we highlighted the importance of ADP-ribose orientation within the binding clef for activity. Moreover, we designed and tested potential inhibitors, identifying key molecular determinants for drug optimisation studies. These studies can lead more broadly to the development of antiviral tools against other human pathogens

Mutations on VEEV nsP1 relate RNA capping efficiency to ribavirin susceptibility

International audienceAlphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes capping of genomic and subgenomic RNAs. The capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7GTP; (ii) guanylylation of the enzyme to form a m7GMP-nsP1adduct; (iii) transfer of m7GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions

Structure and Sequence Requirements for RNA Capping at the Venezuelan Equine Encephalitis Virus RNA 5′ End

International audienceVenezuelian enquine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5′ end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5′-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process

Macro1 domain residue F156: A hallmark of SARS-CoV-2 de-MARylation specificity

International audienceSARS-CoV-2 is a large, enveloped and positive sense single stranded RNA virus. Its genome codes for 16 non-structural proteins. The largest protein of this complex is nsp3, that contains a well conserved Macro1 domain. Viral Macro domains were shown to bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to protein substrates. They carry ADPribose hydrolase activities implicated in the regulation of innate immunity. SARS-CoV-2 and SARS-CoV show widely different induction and handling of the host interferon response. Herein, we have conducted a mutational study on the key amino-acid residue F156 in SARS-CoV-2, pinpointed by bioinformatic and structural studies, and its cognate residue N157 in SARS-CoV. Our data suggest that the exchange of these residues slightly modifies ADP-ribose binding, but drastically impacts de-MARylation activity. Alanine substitutions at this position hampers PAR binding, abolishes MAR hydrolysis of SARS-CoV-2, and reduces by 70% this activity in the case of SARS-CoV