Site-Specific Labelling of the Flavivirus Envelope Protein using Genetic Code Expansion

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Larman, Bridget

Advisors dc:contributor.advisor

• Modis, Yorgo
• Pierson, Theodore

• Flavivirus
• Zika virus
• Antibody-mediated immunity
• Mechanism of neutralisation
• Viral conformational dynamics
• Genetic code expansion
• Amber suppression
• Non-canonical amino acids

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/95881804-4a8f-4cec-9cf0-68ecaffd3a5e/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.116468

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/381157