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oai:www.repository.cam.ac.uk:1810/3811572025-03-08T01:41:38Zcom_1810_219479com_1810_34581col_1810_219488

Site-Specific Labelling of the Flavivirus Envelope Protein using Genetic Code Expansion https://doi.org/10.17863/CAM.116468 Larman, Bridget Modis, Yorgo Pierson, Theodore Flaviviruses are arthropod-borne viruses that constitute a significant global public health burden. The envelope protein (E) orchestrates the processes of virus assembly, budding, and entry into cells and is the primary target of neutralising antibodies. The E proteins arranged on the surface of the virion are not static and pose a dynamic landscape for recognition by host factors, including antibodies. Many antibodies bind virion surfaces not predicted to be accessible using static models, yet neutralise infection by binding epitopes exposed in the structural ensemble of states sampled by the virion. Understanding how neutralising antibodies bind these E protein epitopes to prevent infection is vital for developing vaccines and evaluating potential therapeutic antibodies. In this work, I employed reporter virus particle technology to explore the mechanism of action of novel monoclonal antibodies that neutralised various flaviviruses, including Zika virus (ZIKV). Additionally, this work aimed to expand understanding of the epitopes exposed by the dynamic ZIKV particle. With this goal, I applied genetic code expansion to incorporate the non-canonical amino acid BCN-K at specific sites in the E protein. BCN-K is a versatile non-canonical amino acid that can be labelled via reactions with both tetrazine- and azide-conjugated probes. I demonstrated that BCN-K could be incorporated at a range of sites in soluble recombinant E and intact virions and that these proteins could be site-specifically labelled with fluorophores and biotin. These techniques will advance efforts to identify ZIKV-specific B cells for antibody discovery. Additionally, I explored how different labelling approaches might be used to probe dimer stability and the conformational dynamics of the virion. This work lays the foundation for novel approaches using genetic code expansion and site-specifically labelled particles to explore many aspects of flavivirus biology. University of Cambridge 2024-10-02 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/381157 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2f703edd-5e12-493d-825a-7ba1f9dfea6c/download 3d59e3faffdf70b8f26ff65e31be8624 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/95881804-4a8f-4cec-9cf0-68ecaffd3a5e/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Flavivirus Zika virus Antibody-mediated immunity Mechanism of neutralisation Viral conformational dynamics Genetic code expansion Amber suppression Non-canonical amino acids