Bamford, Rebecca;
(2022)
Investigating the role of myristoylation in vaccinia virus membrane fusion.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The vaccinia virus (VACV) entry-fusion complex (EFC) consists of 11 proteins which are required to mediate membrane fusion. Whilst it is known that repression of individual proteins disrupts EFC polarisation and abrogates fusion, a detailed understanding of the fusion mechanism remains unclear. Amongst the 11 EFC proteins, 3 (A16, G9 and L1) are modified by myristoylation, a covalent lipid modification with roles in many biological processes, including cellular signalling and subcellular targeting. This thesis investigates the role of myristoylation in VACV membrane fusion by generating and characterising a myristate-null VACV. While myristate-null virions appeared structurally normal, analysis of the VACV fusion machinery by super-resolution microscopy revealed that the EFC was depolarised on individual virions. Consistent with this defect, virions produced in the absence of myristoylation showed severely impaired membrane fusion activity and a significant reduction in specific infectivity. While an L1 myristate-null mutant could not be generated, mutation of the myristoylation sites in A16 and G9, individually and in combination was possible. Investigation of the early stages of VACV’s lifecycle indicated that both A16 and G9 myristoylation is important for virus lipid mixing and the rate of early gene expression with the A16/G9 double mutant being most impaired. These results highlight the redundancy, and demonstrate the importance, of EFC protein myristoylation to assure rapid and efficient membrane fusion during VACV entry. Additionally, this thesis provides insight into the spatial-temporal properties of EFC proteins during VACV morphogenesis. EFC proteins could only insert into the viral membrane following removal of the D13 scaffold and cleavage of proteins that are proteolytically processed during the IV-to-MV transition. This indicates that the EFC assembles and polarises within the mature virion membrane.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Investigating the role of myristoylation in vaccinia virus membrane fusion |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10143103 |
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