The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

International audienceViral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltrans-ferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conforma-tions during RNA synthesis

Characterization of capping enzyme of West Nile Virus and human metapneumovirus

Ma thèse a porté sur l’étude des activités enzymatiques impliquées dans la formation de la coiffe de deux virus à ARN: le virus du Nil Occidental (WNV) et le métapneumovirus humain (hMPV). Ces virus codent pour des enzymes assurant l’ajout de la coiffe de type-1 (m7GpppN2’Om) à l’extrémité 5’ de leur ARNm.Le domaine N-terminal de la protéine NS5 (NS5MTase) du WNV porte les activités N7- et 2’O-méthyltransférases (N7- et 2’O-MTases) et il a été proposé que NS5MTase puisse également porter l’activité guanylyltransférase (GTase). J’ai identifié in vitro des résidus clés impliqués dans l’interaction entre NS5MTase et des ARN substrats de chaque activité MTase. Nos résultats démontrent que le site de fixation de la coiffe est nécessaire lors de la 2’O-méthylation et ne l’est pas pour la N7-méthylation. En parallèle, j’ai recherché des résidus catalytiques de la GTase par la méthode de génétique inverse. Des résultats préliminaires indiquent que la mutation K29A induit un défaut de réplication. Ce résidu pourrait donc être impliqué dans l’activité GTase de NS5MTase.Concernant hMPV, j’ai effectué une analyse fonctionnelle du domaine CR-VI+ de la protéine L. J’ai démontré que CR-VI+ possède les activités N7- et 2’O-MTases et j’ai identifié les résidus impliqués dans le recrutement de l’ARNm. L’ordre de méthylation est non canonique avec la 2’O-méthylation qui précède la N7-méthylation. Enfin, j’ai également démontré que CR-VI+ possède une activité d’hydrolyse du GTP.Ce travail démontre que ces MTases possèdent 2 voire 3 des activités enzymatiques nécessaires à la formation de la coiffe, et représentent donc une cible de choix pour le développement d’inhibiteurs.My PhD project is focus on the study of the enzymatic activities involved in the RNA capping pathway of two RNA viruses: the West Nile Virus (WNV) and the human metapneumovirus (hMPV). These viruses encode for enzymes allowing the addition of a cap-1 structure (m7GpppN2’Om) to their mRNA 5’ ends. The NS5 N-terminal domain (NS5MTase) of WNV harbours the N7- and 2’O-methyltransferase activities (N7- and 2’O-MTase); and it has been proposed that NS5MTase also bears a guanylyltransferase activity (GTase). I have identified residues involved in the NS5MTase interaction sites with their RNAs substrate. My assays demonstrate the importance of the cap-binding site for the 2’O-methylation but not for the N7-methylation. In parallel, I have tried to identify putative catalytic residues of the GTase activity by reverse genetics. Preliminary results suggest that NS5MTase K29 could be a catalytic residue.Concerning hMPV, I performed a functional analysis of CR-VI+ domain of the protein L. I demonstrated that the CR-VI+ domain harbours the N7- and 2’O-MTase activities and identified the residues involved in the mRNA recruitment. I showed that the methylation order is not canonical with the 2’O-methylation preceding the N7-methylation. Finally, I showed that the domain harbours an additional GTP hydrolysis activity, representing the first step of RNA cap formation for Mononegavirales.This work demonstrates that this MTase domains harbour 2 or 3 of the enzymatic activities required for viral RNA cap synthesis and represent attractive targets for the development of antivirals

Les forces et faiblesses du système auditif.

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Is there a relationship between speech identification in noise and categorical perception in children with dyslexia?

Purpose: Children with dyslexia have been suggested to experience deficits in both categorical perception (CP) and speech identification in noise (SIN) perception. However, results regarding both abilities are inconsistent, and the relationship between them is still unclear. Therefore, this study aimed to investigate the relationship between CP and the psychometric function of SIN perception. Method: Sixteen children with dyslexia, 16 chronological-age controls, and 16 reading-level controls were evaluated in CP of a voicing continuum and in consonant identification in both stationary and fluctuating noises. Results: There was a small but significant impairment in speech identification performance of children with dyslexia in stationary noise compared with chronological age– matched controls (but not reading level–matched controls). However, their performance increased in a fluctuating background, hence suggesting normal masking and unmasking effects and preserved sensory processing of speech information. Regarding CP, location of the phoneme boundary differed in the children with dyslexia compared with both control groups. However, scrutinizing individual profiles failed to reveal consistently poor performance in SIN and CP tasks. In addition, there was no significant correlation between CP, SIN perception, and reading scores in the group with dyslexia. Conclusions: The relationship between the SIN deficit and CP, and how they potentially affect reading in children with dyslexia, remains unclear. However, these results are inconsistent with the notion that children with dyslexia suffer from a low-level temporal processing deficit and rather suggest a role of nonsensory (e.g. attentional) factors in their speech perception difficulties.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

MMN and P300 are both modulated by the featured/featureless nature of deviant stimuli

Objective: This study was designed to test the effect of the featured/featureless nature of deviant stimuli on mismatch negativity (MMN), P300 (P3a and P3b) and on behavioral discrimination performances. Methods: Ten healthy adults were submitted to stimuli contrasted by the presence or absence of a frequency sweep. Discrimination performances were collected during the neurophysiological sessions. Results: MMN, P3a and P3b were much larger for featured deviants than for featureless ones. Behavioral data (d', at ceiling level, and reaction times) were not affected by the featured/featureless nature of the deviant stimulus. Conclusion: Behavioral results and MMN amplitudes are in accordance with our previous study, using the same design albeit in an ignore condition and with collection of the behavioral data deferred until after the neurophysiological sessions. The present study strengthens previous evidence suggesting that two mechanisms contribute to the MMN evoked by featured deviants: the memory comparison process and the adaptation/fresh-afferent phenomenons. Significance: We here demonstrate that the neurophysiological processes underlying P300 generation are also impacted by the featured/featureless nature of the deviant stimulus and that the dissociation from behavioral data, which are not impacted, is also observed when both types of data are recorded simultaneously.info:eu-repo/semantics/publishe

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain (vol 42, pg 11642, 2014)

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MMN and P300 are both modulated by the featured/featureless nature of deviant stimuli

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X-ray structure and activities of an essential Mononegavirales L- protein domain

The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site (SAMP) also contains a novel pocket (NSP) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the SAMP-adjoining site holding the nucleotides undergoing methylation (SUBP) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity

X-ray structure and activities of an essential Mononegavirales L-protein domain

International audienceThe L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site (SAMP) also contains a novel pocket (NSP) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the SAMP-adjoining site holding the nucleotides undergoing methylation (SUBP) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2′O and N7 positions, and also displays nucleotide triphosphatase activity