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Structure and mechanism of viral and cellular membrane fusion machineries

Periodic Reporting for period 3 - MEMBRANEFUSION (Structure and mechanism of viral and cellular membrane fusion machineries)

Reporting period: 2018-07-01 to 2019-12-31

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. We are applying a combination of state-of-the-art cryo-electron tomography, image processing and correlative fluorescence and electron microscopy methods to obtain detailed structural information on assembled fusion machineries and of fusion intermediates both in vitro and in vivo. By determining how viral and synaptic 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.

Regulated fusion between biological membranes represents a central function that is essential to life. This project will deliver new knowledge on an important, basic biological process. 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 will deliver data that may be 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 will help develop methods to do this.
We have hired project staff, established experiments, data collection protocols and image analysis pipelines. We have reconstituted membrane fusion in vitro for both influenza virus and synaptic vesicle fusion, and have generated cryo-electron microscopy images that will help us to understand aspects of the mechanism of membrane fusion. We have collected cryo-electron microscopy images of HIV-1 and other viruses from which we are deriving new information on the arrangement of the matrix protein layer that underlies the viral membrane.

We have improved methods for the collection and processing of data from cryo-electron tomography, and have shared these with the community. These methods have allowed new structures to be determined, including that of the nucleocapsid of Ebola virus.
Progress beyond the state of the art that has been published includes improved approaches for the collection and processing of cryo-electron tomography data. We have tested and used these methods to determine the structure of the Ebola virus nucleocapsid.

Until the end of the project, we aim to derive new knowledge of membrane fusion mechanisms, and thereby obtain a deeper understanding of mechanisms of virus infection and of synaptic transmission. We will also improve methods and techniques that can be applied in other research projects.