Etudes multi-échelles de l'assemblage de la nucléocapside du virus de la rougeole

Multi-scale studies of Measeles virus nucleocapsid assembly

• Auteur / Autrice : Serafima Guseva
• Direction : Rob W. H. Ruigrok, Martin Blackledge
• Type : Thèse de doctorat
• Discipline(s) : Biologie structurale et nanobiologie
• Date : Soutenance le 18/05/2021
• Etablissement(s) : Université Grenoble Alpes
• Ecole(s) doctorale(s) : École doctorale chimie et science du vivant (Grenoble ; 199.-....)
• Partenaire(s) de recherche : Laboratoire : Institut de biologie structurale (Grenoble)
• Jury : Président / Présidente : Winfried Weissenhorn
• Jury : Examinateurs / Examinatrices : Rob W. H. Ruigrok, Martin Blackledge, Tanja Mittag
• Jury : Rapporteurs / Rapporteuses : Markus Zweckstetter, Marie-Anne Rameix-Welti

Mots clés

Mots clés contrôlés

Interfaces liquide-liquide

Nucléoprotéines

Virus de la rougeole

Protéines virales

Virologie

Mots clés libres

Nmr

Idp

Virus de la rougeole

Séparation de phases liquide-liquide

Usines virales

Dynamique des protéines

SARS-COV-2

Measeles virus

Nmr

Idp

Liquid-liquid phase separation

Viral factories

Protein dynamics

SARS-COV-2

Résumé

La séparation de phases liquide-liquide est un phénomène crucial dans toute la biologie, impliqué dans de multiples processus cellulaires et responsable de la formation d'organelles sans membrane qui sont essentielles pour l'organisation spatiale et temporelle intracellulaire. Il a été proposé que de tels organites soient impliqués dans la formation des usines de réplication virale, qui résultent de l'infection par un certain nombre de virus à ARN simple brin négatif.Ici, nous étudions la séparation de phase de la machinerie de réplication de la rougeole it in vitro, nous identifions la nature et la localisation des interactions requises et montrons que certains processus essentiels sont accélérés, en particulier l'assemblage de la nucléocapside où la nucléoprotéine de la rougeole se lie à l'ARN génomique pour former des capsides hélicoïdales.Les protéines impliquées dans la réplication de la rougeole sont connues pour être phosphorylées dans la cellule, cependant, le rôle fonctionnel de cette modification post-traductionnelle n'était pas compris auparavant. Au cours de ma thèse, nous avons découvert que la phosphorylation de la phosphoprotéine de la rougeole déclenche l'assemblage de la nucléocapside.Pour mieux comprendre l'origine physique de la séparation de phase des protéines, nous avons étudié la structure et la dynamique des protéines en utilisant un système modèle. En utilisant une partie désordonnée de la nucléoprotéine de la rougeole, nous comparons le comportement de la protéine entre les états dilué et condensé. En utilisant la spectroscopie RMN, nous effectuons une comparaison spécifique au site des amplitudes de mouvement et des échelles de temps de la protéine entre les phases.Enfin, il a également été démontré que le SARS-COV-2 forme des condensats viraux ne nécessitant qu'une seule protéine virale : la nucléoprotéine. Nous caractérisons ici les régions intrinsèquement désordonnées du SARS-COV-2 N et démontrons sa séparation de phase it in vitro.

Phase separation is now considered to be one of the hottest topics in biology which might transform our understanding of biology and biomedicine. Despite multiple examples and undoubtable proofs of their existence in cells, the scientific community still discusses whether it is a "Sloppy science or ground-breaking idea”. Phase separation is responsible for the formation of membaneless organelles which are involved in the arrangement of cell content. The cytoplasm and the nucleus are shown to be full of such structures. And even if several process take place in cytoplasms, they can be spatially separated with membaneless organelles. The discovery of biocondensates in the cell can finally answer the fundamental question of how cells organise the necessary molecules at the right place and time to carry out a particular function.The basics of the phase separation phenomena are still not fully understood especially for biological systems. However, the importance of intrinsically disordered proteins (IDPs) is highlighted in many studies. IDPs role in biology is underestimated in general, while school and university programs mostly ignore their existence.par To understand how membaneless organelles control the cellular functions it is important to define the properties of the system. A range of biophysical techniques can be used to characterise macroscopic features of the droplets. However, NMR is probably the only technique which allows to study phase separation at the molecular level and obtain atomic resolution information about interactions involved in their formation, in terms of dynamic and structural changes associated with the phase transition.One of the examples of phase separation in biology is the formation of viral proteins biocondensates. It appears that viral components are not dissolved in the cytoplasm but concentrated in so-called viral factories. Multiple advantages for such organisation can be found: Increase in the rate of chemical reactions due to the increase of concentration, control of the environmental conditions, protection from the host cell immune system recognition. In this thesis, phase separation phenomena is studied on the basis of measles virus replication machinery. The machinery requires the polymerase and two accessory proteins : Nucleoprotein and Phosphoprotein. The Nucleoprotein binds to the viral RNA and forms nucleocapsids, the Phosphoprotein controls this reaction as well as polymerase activity, formation of viral factories and probably many others which are not identified yet. The Phosphoprotein has multiple binding sites with the Nucleoprotein which are present at different stages of viral cycle. How the Phosphoprotein regulates its interactions with the Nucleoprotein and thus controls viral replication is an open question.The present study is important for understanding the mechanism of measles replication in cell as well as related viruses. And also provides the advances in the description of the basics of liquid-liquid phase separation of proteins thereby linking physics with cellular biology.This thesis is dedicated to several subjects:First we aimed to test the ability of measles virus Nucleoprotein and Phosphoprotein to phase separate and to determine the mechanism of droplet formation. Also, to determine the function of viral biocondensates - this is presented in Chapter 3.In addition, I aimed to study phosphorylation of measles Phosphoprotein it in vitro and understand the functional role of phosphorylation - this is the subject of Chapter 4.To better understand the physical origin of protein phase separation, we studied protein structure and dynamics using a model system. Using a disordered part of measles nucleoprotein, we compare protein behaviour between dilute and condensed states. Using NMR spectroscopy, we perform a site-specific comparison of motional amplitudes and timescales of the protein between phases (Chapter 5).