Fusion of two biological membranes is essential to life. It is required during organism development, for trafficking of material between cellular compartments, for transfer of information across synapses, and for entry of viruses into cells. Fusion must be carefully controlled and the core fusion components are typically found within a complex regulatory machine. The objective of this project was to applying a combination of state-of-the-art imaging techniques, including cryo-electron tomography, computational image processing and correlative fluorescence and electron microscopy, to obtain detailed structural information on assembled fusion machineries and of fusion intermediates both in vitro and in vivo. By determining how viral and cellular fusion complexes reposition and restructure prior to fusion, how they arrange around the fusion site, how they reshape the membrane to induce fusion, and how these processes can be regulated and inhibited, we aim to understand more about the mechanisms of fusion.
Influenza A and HIV-1 are major human pathogens, and the fusion of these viruses with the host cell is a target for drug development and a process interfered with by neutralizing antibodies. By contributing to understanding of these viruses, this project aimed also to provide new knowledge that valuable in the design and development of strategies for disease treatment or prevention.
It is challenging to obtain detailed structural information on biological systems in situ within complex environments. This project aimed to develop methods to do this.
During the course of the project we improved methods for cryo-electron tomography and subtomogram averaging – two related techniques that allow imaging proteins, viruses and cellular components in three dimensions. We applied these techniques to study viruses and “test-tube” systems that mimic aspects of viruses and cells. We revealed information on the arrangement of proteins at the fusion sites between vesicles that mimic those occurring at synapses in the brain. We showed how the small proteins that underlie the membrane surface of influenza A, HIV-1 and Ebola virus are arranged and shed light on how they might contribute to virus assembly and change their structure to facilitate virus fusion and entry. In the later stages of the project we determined the structure of the fusion machinery of SARS-CoV-2 from 3D reconstructions of intact virus particles, and generated SAARS-CoV-2 proteins that were used for diagnostic serology assays, for studying the mechanism of fusion, and for attempting to identify new therapeutic routes.
