Highlights

Cryo-EM structure of a key enzyme in action gives insights into the replication of a human pathogenic virus

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening human pathogens for which there is currently no treatment (La Crosse virus, Hantaan virus, Crimean Congo virus, Lassa virus). The replication and transcription of their genome are essential steps of their viral cycle and are catalyzed by a key viral enzyme : the RNA-dependent RNA polymerase. The MEM group at IBS, in collaboration with Dr. Cusack’s group at EMBL Grenoble, describes here the structure of the complete RNA-polymerase of the La Crosse virus obtained at 3 Å resolution by cryo electron microscopy, using data collected on the Glacios cryo-microscopes from IBS and Krios from ESRF. This structure reveals the position and organization of the C-terminal part of the RNA polymerase which includes a cap-binding domain necessary for transcription initiation. Two states could be visualized, pre-initiation and elongation. In particular, this allows to highlight the conformational changes necessary for the formation of a double-stranded 10-base pair RNA in the active site cavity during elongation. The structural details and dynamics of the functional elements identified provide mechanistic insight into bunyavirus transcription and may be crucial for the future development of RNA polymerase inhibitors.

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational change. Benoît Arragain, Grégory Effantin, Piotr Gerlach , Juan Reguera , Guy Schoehn, Stephen Cusack, Hélène Malet. Nature Communications 2020 ;11(1):3590. doi : 10.1038/s41467-020-17349-4.

Contact : Hélène Malet

Following protein aggregation in real time by neutron spectroscopy

Protein aggregation into amyloid superstructures is the molecular manifestation of a large variety of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Evidence is increasing that transient on-pathway oligomers are actually the toxic species, such that time-resolved monitoring of protein aggregation is highly desirable. Whereas time-resolved structural techniques have been developed and applied to study protein aggregation, methods accessing protein center-of-mass and internal diffusion dynamics remain only sparsely available. Yet, changes in protein dynamics have been postulated to play an essential role in driving and accompanying protein aggregation.
Scientists of the Institut Laue Langevin, the Institut de Biologie Structurale and the University of Copenhagen developed a time-resolved version of incoherent neutron scattering experiments on IN16B to follow protein aggregation and applied it to study the assembly of lysozyme in aqueous solution into particulate superstructures. Surprisingly, the internal protein dynamics on the nano-to picosecond time scale does not change during the entire aggregation process. The center-of-mass diffusion, on the other hand, decreases as aggregation proceeds and can be well explained by a single exponential process. By complementing the neutron results with fluorescence measurements, electron microscopy, infrared spectroscopy, x-ray powder diffraction, and dynamic light scattering, a comprehensive picture was painted in which lysozyme particulate formation is a one-step process with protein backbone and side chain dynamics remaining unchanged throughout aggregation.
The work establishes a framework to follow protein aggregation quantitatively in real time at a molecular level, simultaneously accessing center-of-mass and internal diffusivities, which will be invaluable for addressing pathological pathways of protein aggregation. This framework is not limited to proteins, but can be applied to macromolecules in general to study a large variety of processes such as self-assembly and emerging aggregates and crystals.

Tracking Internal and Global Diffusive Dynamics During Protein Aggregation by High-Resolution Neutron Spectroscopy. Pounot K, Chaaban H, Foderà V, Schirò G, Weik M, Seydel T. J.Phys.Chem.Lett. 11, 15 (2020)

Contact : Martin Weik

Hélène Malet appointed Junior Member of the Institut Universitaire de France

Hélène Malet, Associate Professor at the University Grenoble Alpes and research scientist in Dr. Guy Schoehn’s Methods and Electron Microscopy group at IBS, is appointed Junior Member of the Institut Universitaire de France (IUF) from October 1, 2020 for a period of five years.

Viral replication and transcription are key steps of the viral cycle. Hélène Malet analyses the structure of viral proteins involved in these processes, in particular viral polymerases. During her thesis, carried out under the supervision of Dr. Bruno Canard at the AFMB, Marseille, she characterized by X-ray crystallography a polymerase structure of the Flaviviridae family, to which the Dengue virus belongs. Then, eager to learn a complementary method of structural biology, she did a post-doctorate in electron microscopy in the laboratory of Pr. Helen Saibil at Birkbeck College, London. Since then, she combines her interest in electron microscopy and viral replication. She undertook a post-doctoral fellowship on the structural analysis of Peribunyaviridae polymerase in the group of Dr. Stephen Cusack at EMBL Grenoble, before being recruited as an UGA Associate Professor at IBS in the team of Dr. Guy Schoehn in the Electron Microscopy and Methods group.

Her research project focuses on the structural and functional analysis of bunyavirus replication, a viral order consisting of many highly pathogenic human viruses against which no drugs or vaccines are available. The latest advances in electron microscopy and the presence of state-of-the-art electron microscopes at IBS and ESRF enable to determine high-resolution structures of these essential enzymes thereby revealing their modes of action, a key step for the future development of anti-virals. In the longer term, this project aims to understand the mechanisms of interactions between viral proteins and host proteins involved in the regulation of viral replication, combining high-resolution electron microscopy of isolated particles and cellular electron microscopy, allowing an integrative view of these processes. This project is financially supported by the ANR (HiPathBunya) and will make an extensive use of IBS technology platforms managed by ISBG and funded by FRISBI and Gral. The appointment at the IUF will allow her to devote more time to this project.