Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

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

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).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

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

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).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

Multi-scale studies of Measeles virus nucleocapsid assembly

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)

The Nucleoprotein and Phosphoprotein of Measles Virus

International audienceMeasles virus is a negative strand virus and the genomic and antigenomic RNA binds to the nucleoprotein (N), assembling into a helical nucleocapsid. The polymerase complex comprises two proteins, the Large protein (L), that both polymerizes RNA and caps the mRNA, and the phosphoprotein (P) that co-localizes with L on the nucleocapsid. This review presents recent results about N and P, in particular concerning their intrinsically disordered domains. N is a protein of 525 residues with a 120 amino acid disordered C-terminal domain, Ntail. The first 50 residues of Ntail extricate the disordered chain from the nucleocapsid, thereby loosening the otherwise rigid structure, and the C-terminus contains a linear motif that binds P. Recent results show how the 5' end of the viral RNA binds to N within the nucleocapsid and also show that the bases at the 3' end of the RNA are rather accessible to the viral polymerase. P is a tetramer and most of the protein is disordered; comprising 507 residues of which around 380 are disordered. The first 37 residues of P bind N, chaperoning against non-specific interaction with cellular RNA, while a second interaction site, around residue 200 also binds N. In addition, there is another interaction between C-terminal domain of P (XD) and Ntail. These results allow us to propose a new model of how the polymerase binds to the nucleocapsid and suggests a mechanism for initiation of transcription

Structure, dynamics and phase separation of measles virus RNA replication machinery

International audienceThe measles virus replication complex represents a potentially important, but as yet relatively unexplored target for viral inhibition. Little is known about the molecular mechanisms that underpin replication and transcription in paramyxoviruses. In recent years it has become clear that conformational dynamics play an important role in paramyxoviral replication, and that a complete understanding of the viral cycle requires a description of the structural plasticity of the different components. Here, we review recent progress in this direction, covering the dynamics of the nucleocapsid assembly process, high resolution structure and dynamics of protein:RNA interactions, and the investigation of the role of intrinsic conformational disorder in pre-assembly nucleoprotein/phosphoprotein complexes. Finally, we discuss the role of viral factories in the form of phase-separated membraneless organelles formed by measles virus phospho and nucleoproteins that promote the assembly of nucleocapsid structures

1^1H, 13^{13}C and 15^{15}N Backbone chemical shift assignments of the n-terminal and central intrinsically disordered domains of SARS-CoV-2 nucleoprotein

International audienceThe nucleoprotein (N) from SARS-CoV-2 is an essential cofactor of the viral replication transcription complex and as such represents an important target for viral inhibition. It has also been shown to colocalize to the transcriptase-replicase complex, where many copies of N decorate the viral genome, thereby protecting it from the host immune system. N has also been shown to phase separate upon interaction with viral RNA. N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45$-$175 and 264$-$365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1$-$44 and N3, spanning residues 176$-$263). Our assignment provides the basis for the identification of inhibitors and functional and interaction studies of this essential protein

Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

International audienceMany viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. We demonstrate that purified nucleoproteins (N) and phosphoproteins (P) of measles virus form liquid-like membraneless organelles upon mixing in vitro. We identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows us to follow the modulation of the dynamics of N and P upon droplet formation, while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in measles virus replication

NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins

International audienceIntrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health