Using electron microscopy to understand viral infection and control: structural studies of murine norovirus and a novel vaccine platform technology

Viruses have unquestionable impact on our societies. While much progress has been made in our understanding of viral disease, we remain vulnerable, and the continual risk of emerging viral pathogens highlights the importance of continued and more detailed study. Through structural investigations of an infectious virus and a vaccine platform technology, this thesis illustrates how cryogenic electron microscopy (cryoEM) can contribute to understanding and controlling viral infection. Firstly, using murine norovirus (MNV) as a model system, cryoEM was applied to the study of noroviruses, the most common cause of acute non-bacterial gastroenteritis. Structures were determined for a number of MNV variants, including a thermally stabilised mutant with potential applications to vaccine design. This shed insight into the dynamic nature of MNV capsids, and the importance of this for viral infectivity and stability. Subsequent studies of MNV produced in an alternative cell line revealed a rigidified capsid, provoking questions around the influence of expression system on structure, and the implications for comparative structural analysis. Finally, the molecular interactions of MNV with a bile acid were investigated, and complex image processing techniques were applied to probe non-capsid components of the virion, including the viral genome. Secondly, structural investigations were carried out into a novel vaccine platform technology. Termed ‘VelcroVax’, the technology comprises virus-like particles formed from hepatitis B virus core antigen, modified to display Affimers for the capture and presentation of target antigens. CryoEM was used to determine the structures of different prototype constructs, including one displaying a viral antigen, Junín virus glycoprotein 1. Based on analysis of these structures, recommendations are made for improving the design and expanding the functionality of VelcroVax constructs. Finally, the importance of considering contaminants within cryoEM datasets is illustrated through structural determination of a yeast fatty acid synthase identified in a VelcroVax dataset.