PHD2022-13
Physical and structural characterization of the interplay between the cell membranes, actin filaments and Chikungunya protein nsP1 ruling the formation of viral organelles in the host cell
Host laboratory and collaborators
Juan REGUERA (AFMB) / juan.reguera@univ-amu.fr
Ignacio CASUSO (INSERM) / ignacio.casuso@inserm.fr
Abstract
The alphaviral protein nsP1 rearranges the cellular cytoskeleton and takes over the membranes as a first step for the generation of viral membranous organelles. nsP1 membrane binding results in the nsP1 oligomerisation in dodecameric pores which activates its capping activity, essential for viral replication and transcription. This is an essential process where membrane binding, membrane bending, protein oligomerisation and allosteric activation of the enzyme are coupled. We have preliminary data showing nsP1 bending membranes by AFM and binding actin filaments by sedimentation and electron microscopy. This thesis aims to explore the forces, structures and dynamics ruling viral organelle formation by the interplay of actin, cell membranes and nsP1. We will provide a model for understanding the formation of cell membranous organelles.
Keywords
Viral infection, Alphavirus, membrane remodeling, High-speed atomic force microscopy, Cryo-electron microscopy, Biophysics, cytoskeleton.
Objectives
The objectives are to: i) Study the affinities and conformational changes of the membrane-bound nsP1 upon interaction with actin filaments and its effect on actin filaments growth. ii) Determine the structure, their dynamical transitions and their energetics of these assemblies at atomic, molecular and supramolecular levels. iii) Define the biophysical mechanisms of the action of the nsP1 and actin complexes on model membranes using molecular-level data correlated to observations in infected cells of the cell membrane remodeling.
Proposed approach (experimental / theoretical / computational)
Visualization of the monomeric and oligomeric nsP1 binding and action on membranes will be attained by High-Speed AFM at the laboratory of I. Casuso (LAI). Live during the HS-AFM video imaging, actin and nsP1 will be added to the system, all the changes in the membrane structure and protein dynamics will be recorded. Such type information will be critical for establishing the biomolecular physics underlying viral membrane remodeling. The interactions of actin and nsP1 observed by HS-AFM will be reproduced in cryo-EM grids for the high-resolution determination of these structures by cryo-electron microscopy in J. Reguera’s team (AFMB). The combination of structures with dynamics of nsP1, actin oligomerisation, actin-nsP1 interactions and membrane bending will allow to propose an accurate model explaining the membrane reconfigurations observed in infected cells.
Interdisciplinarity
The thesis will combine the latest techniques on structural biology, high-speed atomic force microscopy and fluorescence microscopy. By combining these techniques, we will have the opportunity to understand from the atom to the supramolecular assembly to the cell the physics, structures and dynamics of the re-configuration of cellular membranes for setting up a particular infection environment: This process necessarily involves the interplay of essential viral and cellular factors as are, in this particular case, nsP1, acting filaments and cell membranes. The data expected will elucidate the physical forces needed for these cell transformations, the structural factors allowing these forces, a statistical comprehension of events of protein-protein and membrane-protein interactions and model, from this data, how the global reconfiguration of the cell membranes may occur.
Expected profile
The candidate should be a motivated and ambitious. The candidate should fill as good as possible the following requirements: i)Background in biology, chemistry, pharmacology or physics. ii) Research skills (if possible on molecular imaging by atomic force microscopy and/or microbiology). iii) Ability in reading, analyzing and writing scientific literature and iv) Publication record (although not compulsory).
Is this project the continuation of an existing project or an entirely new one? In the case of an existing project, please explain the links between the two projects
Yes, this project is the continuation of an existing project on the characterization of alphavirus replication complexes that was financed by the ATIP-Avenir program and the Fondation Bettencourt Shueller to Juan Reguera. Our latest results show the nsP1 protein of the alphavirus replication complexes assembled in a dodecameric monotopic membrane pore. The objective now is to study the interplay of actin, membranes and nsP1 on the assembly of viral replication factories.
2 to 5 references related to the project
1: Jones R, Bragagnolo G, Arranz R, Reguera J. Capping pores of alphavirus nsP1 gate membranous viral replication factories. Nature. 2020 Dec 16:1–5. doi: 10.1038/s41586-020-3036-8. Epub ahead of print. PMID: 33328629; PMCID: PMC7739802.
2: Casuso I, Redondo-Morata L, Rico F. Biological physics by high-speed atomic force microscopy. Philos Trans A Math Phys Eng Sci. 2020 Dec 11;378(2186):20190604. doi: 10.1098/rsta.2019.0604. Epub 2020 Oct 26. PMID: 33100165; PMCID: PMC7661283.
3: Zuttion F, Redondo-Morata L, Marchesi A, Casuso I. High-Resolution and High-Speed Atomic Force Microscope Imaging. Methods Mol Biol. 2018;1814:181-200. doi: 10.1007/978-1-4939-8591-3_11. PMID: 29956233.
3 main publications from each PI over the last 5 years
Juan Reguera
1: Jones R, Bragagnolo G, Arranz R, Reguera J. Capping pores of alphavirus nsP1 gate membranous viral replication factories. Nature. 2020 Dec 16:1–5. doi: 10.1038/s41586-020-3036-8. Epub ahead of print. PMID: 33328629; PMCID: PMC7739802.
2: Jones R, Lessoued S, Meier K, Devignot S, Barata-Garcia S, Mate M, Bragagnolo G, Weber F, Rosenthal M, Reguera J. Structure and function of the Toscana virus cap-snatching endonuclease. Nucleic Acids Res. 2019 Nov 18;47(20):10914-10930. doi: 10.1093/nar/gkz838. PMID: 31584100; PMCID: PMC6847833.
3: Gerlach P, Malet H, Cusack S, Reguera J. Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter. Cell. 2015 Jun 4;161(6):1267-79. doi: 10.1016/j.cell.2015.05.006. Epub 2015 May 21. PMID: 26004069; PMCID: PMC4459711.
Ignacio Casuso
1: Zuttion F, Colom A, Matile S, Farago D, Pompeo F, Kokavecz J, Galinier A, Sturgis J, Casuso I. High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action. Nat Commun. 2020 Dec 9;11(1):6312. doi: 10.1038/s41467-020-19710-z. PMID: 33298927; PMCID: PMC7725780.
2: Casuso I, Redondo-Morata L, Rico F. Biological physics by high-speed atomic force microscopy. Philos Trans A Math Phys Eng Sci. 2020 Dec 11;378(2186):20190604. doi: 10.1098/rsta.2019.0604. Epub 2020 Oct 26. PMID: 33100165; PMCID: PMC7661283.
3: Zuttion F, Redondo-Morata L, Marchesi A, Casuso I. High-Resolution and High-Speed Atomic Force Microscope Imaging. Methods Mol Biol. 2018;1814:181-200. doi: 10.1007/978-1-4939-8591-3_11. PMID: 29956233.