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Final Report Summary - FLUMABUD (The role of the influenza virus matrix protein M1 in budding adn virus release)

The long-known flu disease remains a major concern in in the 21th century with the threat of new pandemics associated with the emergence of new strains such as the avian strain H5N1 in the 90’ and more recently the spread of the “pig flue”. The flue is caused by the well-known viral pathogen Influenza. In spite of the efforts and progress reported over the last two decades in elucidating the virus behavior, controversies still exist and the complex processes of its life cycle is far from being completely understood.
The aim of the FLUMABUD project was to use advanced biophysical tools to study the mechanisms by which Influenza C exits its host cell. Here we focus on Influenza’s l budding. During budding, the viral components are recruited at the inner leaflet of the plasma membrane of an infected cell and the membrane is deformed into a bud before pinching-off after membrane fission. Our goal was to investigate the role of the viral matrix protein, M1, a protein connecting genetic material with the virus membrane, in the process. We asked whether the protein alone is capable of deforming lipid membranes into tubular or spherical structures and whether this protein can facilitate membrane abscission, possibly by constricting the bud’s neck. Our approach is widely based on the use of minimal models of the cell membrane to mimic the membrane’s physical environment and membrane properties in a well-controlled in vitro experimental environment. Such mimics are produced by lipid self-assembly in aqueous solution.
In a first aspect of the project, supported lipid bilayers, i.e. lipid bilayer structures deposited onto a solid support were used in conjunction with surface-sensitive techniques, to gain detailed insights into the binding characteristics of M1. Quartz-crystal microbalance, a label-free biosensing method, was used to probe interaction kinetics and structural properties of protein adlayers. These experiments reveal that initial recruitment to the lipid membranes containing negatively charged lipids is widely mediated by electrostatic interactions but followed by strengthening of the interaction though other forces, as verified by experiments under high ionic strength conditions. This strengthening was hypothesized to be due to protein-protein interactions occurring within the membrane-bound proteins. Experiments performed with a protein truncated on its C-terminal (M1N) suggest that these protein-protein interactions are mediated by the C-terminal end, as M1N adlayers were found to be significantly less stable than the full length ones. A combination of total internal fluorescence microscopy, surface enhance ellipsometric contrast and atomic force microscopy imaging further reveals that the protein forms large scale clusters when in contact with the supported lipid bilayers, indicative of membrane-induced polymerization. Negatively charged DOPS lipids were enriched under the protein clusters and these clusters were found to be ~12 nm in height which corresponds to the height of two M1 molecules.1
A second aspect of the project was devoted to the study of M1-induced membrane deformations and M1-induced fission using giant unilamellar vesicles (GUVs) and confocal microscopy. Here, GUVs of two different lipid compositions. The first composition consisted of equal amounts of the negatively charged lipid DOPS, DOPE and DOPC. The second composition was more native-like as it consisted of lipids extracted from a natural tissue (porcine brain extract), which contains approximately 10% PS lipids, to which 5% PI(4,5)P2 was added. In all cases, addition of M1 to the GUVs resulted in the spontaneous formation of inward tubules into the GUVs although tubulation was more pronounced and required less protein in solution for the native lipid composition. The tubules were rather stiff, had diameters that resembled the one of the native virus and could have length up to micrometers. This observation indicates that M1 alone, i.e. without help of any additional cellular factor is capable of deforming lipid membranes into buds. Experiments with M1N further confirm that the C-terminal domain of M1-C is required, corroborating its essential role for M1-C polymerization on membranes. Our results indicate that M1-C polymerization on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress. Interestingly, for the native composition, M1 was found to assemble into extensive network structures on the surface of GUVs, possibly correlated with partial membrane deformations. At this stage, it is unclear whether the network-like morphology represents a precursor state in the tubulation process or whether it is an alternative dead-end polymerization state, not relevant for the tubulation process. The exact mechanisms leading to tubulation will be the subject of further investigation. In no case membrane fission was observed, indicating that additional cellular or viral factors are needed to pinch off the membrane. 2
In our recent publication 2, our in vitro reconstitution experiments were further combined with structural information on M1 performed by our collaborator group in Grenoble (W. Weissenhorn). Indeed, we present the crystal structure of the N-terminal domain of M1-C and M1-A and show that in spite of low sequence homology both proteins exhibit very similar structures indicating that both proteins may be characterized by a similar structure-function relationship. Small Angle X-Ray Scattering analysis further revealed that that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers as further shown by electron microscopy. This protein assembly is likely to play a key role in stablilizing the virion’s morphology.
In a last assay configuration, attempts were made at reproducing the geometry of the membrane bud by nanotube pulling experiments. In our assay setup, a GUV is immobilized into a micropipette which makes it possible to control the tension on the membrane, by aspiration. A nanotube is pulled from the GUV by help of a bead bound to the GUV and trapped by an optical tweezer. 3 The advantage of this configuration is that the tube diameter can be tuned by controlling the tension applied to the GUV. This setup therefore makes it possible to probe the curvature-dependent binding behaviors. It can further be used to quantify the mechanical action of proteins on the membrane by measuring the changes in the tube pulling forces in the tube. Preliminary experiments reveal that the protein can mechanically stabilize the membrane even if added to the outside of the tubes indicating a scaffolding effect of the protein. Efforts were further directed at more closely reproducing the budding geometry by encapsulating the protein into the vesicle prior to tube pulling. Encapsulation turned out however to be extremely challenging but first step towards a successful encapsulation strategy were eventually achieved. Due to time constraints no additional tube pulling experiments on GUV-encapsulated M1 could be carried out.
In conclusion the FLUMABUD project generated important mechanistic insights into the processes leading to Influenza C budding. Such insights are interesting from a fundamental biological point of view but also essential in the context of the development of new drugs and vaccines required to fight Influenza epidemics and to win the race against a fast evolving pathogen.

1.Ecklund, B.; Rupert, D. L. M.; Radzimanowski, J.; Zahn, R.; Weissenhorn, W.; Bassereau, P.; Bally, M., Assembly and disassembly of the influenza C matrix protein layer on a lipid membrane. in preparation
2.Saletti, D.; Radzimanowski, J.; Effantin, G.; Midtvedt, D.; Mangenot, S.; Weissenhorn, W.; Bassereau, P.; Bally, M., The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model. Sci Rep 2017, 7, 40801.
3.Sorre, B.; Callan-Jones, A.; Manneville, J. B.; Nassoy, P.; Joanny, J. F.; Prost, J.; Goud, B.; Bassereau, P., Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (14), 5622-6.

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