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Structural Studies on Influenzavirus Fusion

Final Report Summary - INFLUENZA FUSION (Structural Studies on Influenzavirus Fusion)

This report describes the results from a Marie Curie Intra-European Fellowship. The work was carried out at the National Institute for Medical Research, Mill Hill, London, UK, which is now the Francis Crick Institute Mill Hill Laboratory. The project achieved both its scientific and training objectives, enabling the fellow to establish her independent research career. Therefore, the fellow had to terminate the fellowship after 15 months to move to the Max Planck Institute for Colloids and Interfaces, Berlin, Germany as a group leader.
The fusion of biological membranes underlies basic cellular processes of vesicular transport and exocytosis, but also the infectivity of enveloped viruses. Cellular and viral membrane fusion is tightly regulated and mediated by fusion proteins, whose mechanism is a topic of intense research. The haemagglutinin (HA) glycoprotein of influenza viruses mediates fusion between viral and cellular membranes at the pH of the endosome to deliver the viral genome into the cytosol. HA is activated by cleavage into the receptor-binding HA1 and the fusion-mediating HA2, which has two membrane-interacting elements: the N-terminal fusion peptide and a C-terminal transmembrane helix. At low pH, HA2 undergoes a series of conformational changes that project the fusion peptide out of a protein pocket towards the target membrane, and subsequently bring the fusion peptide and the transmembrane helix, and likely the two membranes, into close proximity. The conformational changes are defined by crystal structures at neutral and low pH, but the membrane-associated parts of the protein are absent from the structures. The fusion machinery of HA is moreover a prime target for broadly neutralizing antibodies against influenza virus because the fusion domain is much more conserved between different serotypes than other parts of the HA surface. These broadly neutralizing antibodies are pharmaceutical targets as influenza therapeutics and for vaccine design, but the molecular determinants of their broad range are not understood.
This project aimed at providing structural information on the membrane-associated parts of HA before and after membrane fusion by x-ray crystallography. Relatedly, the project also investigated antibodies against the fusion domain of HA, to provide the structural basis of their broad neutralization range. The fellow purified large amounts of different full-length HA proteins in detergent and characterized them with biophysical methods. Crystallization trials for structure determination were ongoing at the end of the fellowship, and are continued in the laboratory. Antibodies against the fusion machinery obtained through collaborations were investigated and characterized by the fellow by biophysical methods, virological assays and electron microscopy.
The fellow solved a high resolution crystal structure of a broadly neutralizing antibody that recognizes the fusion machinery of all influenza A subtypes. In addition, she solved structures of this antibody in complex with both group 1 and group 2 HA molecules at 3.7 and 3.75 Å resolution. The results detail the highly conserved epitope of the antibody, which is distinct from those of other structurally characterized cross-neutralizing antibodies. The CDR loops of the antibody undergo an induced fit movement in order to bind to the epitope. The structures suggest that the antibody prevents both the fusion-inducing conformational change of HA as well as its proteolytic activation, and these suggestions were proven by collaboration partners. The structures show that the locations and orientations of the antibodies bound to group 1 and group 2 HAs are very similar, and suggest that the structurally conserved ability to interact with HA results in effective cross-reactivity. Finally, the structures highlight a peptide motif that seems to be central to cross-group reactivity, which might be used in the preparation of immunogens.
In summary, the results of this project help developing broadly neutralizing antibodies as influenza therapeutics and pave the way to understand membrane fusion.
Concerning the planned training activities, the fellow obtained valuable new skills in membrane protein science, electron microscopy and virology. In addition, she learned important skills in project management, collaboration and communication, and gained new scientific contacts. Ultimately, the training enabled her to obtain a position as a junior group leader and to secure startup funding for her independent lab.