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Structure and mechanism of viral and cellular membrane fusion machineries

Periodic Reporting for period 4 - MEMBRANEFUSION (Structure and mechanism of viral and cellular membrane fusion machineries)

Reporting period: 2020-01-01 to 2021-08-31

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. 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.
Highlights of the project are briefly described here.

The transfer of signals across synapses in the brain requires coordinated fusion of vesicles with the synaptic membrane, and this is controlled by SNARE proteins. Using a system in which the basic parts of this process are reconstituted in the test tube we imaged fusion sites under different conditions and found that the “primed” state, ready for fusion, can have a variable arrangement of proteins.

We developed a correlative microscopy approach that allowed us to identify intermediate stages in influenza fusion using a fluorescent signal and then take images of the same intermediates using cryo-electron tomography.

The most abundant protein in influenza virions is the matrix protein M1, but it was not known how this protein is structured or how it is arranged inside virus particles. We applied a combination of “test-tube” studies with purified proteins, and imaging of intact viruses and virus-like particles using cryo-electron microscopy. For the first time we were able to reveal the complete structure of the protein and see how it is arranged inside virus particles. This suggested how interactions between M1 proteins might help filamentous influenza particles to assemble, and revealed a small cluster of amino acids that might help switch the structure of the protein when the virus enters a new cell. Using a similar approach we also described the arrangement of the matrix protein VP40 in Ebola virus particles.

The matrix protein, MA, of HIV-1 was known to be responsible for recruiting the components of HIV-1 to the site of virus assembly, and to help incorporate the viral fusion protein. We determined the structure and arrangement of MA in immature (as released from cells) and mature (ready to enter a new cell) HIV-1 particles, finding that in both cases MA assembles a regular lattice of protein but that these two lattices are different. The change in MA arrangement correlates with the virus becoming able to fuse with a new cell. Strikingly the change also appears to trigger a change in the lipid bilayer of the virus. These results suggest that rearrangement of MA is a critical aspect of HIV-1 maturation and that MA may have an undescribed role during entry of HIV-1 into a target cell.

We optimized methods and computer scrips to allow more efficient collection and processing of data for obtaining high resolution structural data using cryo-electron tomography. These developments were incorporated into software tools for others to use.

The start of the final reporting period coincided with the start of the SARS-CoV-2 pandemic, and we redirected efforts to study the fusion machinery of SARS-CoV-2 (the spike or S protein). Using expertise gained during the project we were able to quickly produce SARS-CoV-2 S protein (the fusion machinery), and supply this protein to collaborators. This protein was used to develop the first UK accredited serological test for SARS-CoV-2 antibodies as well as to run thousands of tests on healthcare workers.

Using the image processing software developed in the project and the experience of in “in virus” structural studies, we generated three dimensional reconstructions of individual SARS-CoV-2 virions. From these reconstructions, we determined the structure, distribution and the flexibility of S protein on intact virions.

The S protein undergoes major changes in structure before and during cell entry. We also made S proteins that were blocked in particular structural states which we then used to understand more about the structural dynamics of the fusion mechanism, and as antigens to raise or screen for antibodies with particular properties. We also shared these proteins with other researchers designing antiviral compounds or antibodies.
The project is complete

Our three studies on the matrix proteins of HIV-1, influenza A and Ebola virus, together have greatly improved our understanding of how these small but essential proteins are arranged within virus particles, providing a basis for us and other researchers to interpret and design experiments to further study these proteins and how they regulate virus functions including fusion and entry.

Our work on SARS-CoV-2 provided the first high-resolution structural description of the SARS-CoV-2 fusion machinery in place on the surface of the virus particle.
SARS-CoV-2 virus particle