Seeing, Following and Understanding Viruses Via Unnatural Protein Creation
Supervisor: Dr Stephen Carter, School of Infection and Immunity
As well as being major causes of human disease, viruses provide powerful tools for exploring the molecular biology of infected cells. Studying the interactions between a human virus and its host cell exposes the molecular machinery of the cell to detailed study in a way that can have major impacts on health, while the ability to genetically engineer viruses creates powerful precision tools for tracking and capturing molecular interactions. A recent example of this is the ability to engineer viruses such that their proteins contain unnatural amino acids (uAAs), which can be used as a platform for cutting-edge imaging and interaction studies1. This PhD will allow you to apply cutting-edge technologies present at the MRC-University of Glasgow Centre for Virus Research to viruses through a collaboration with the Rosalind Franklin Institute in Oxford.
In this PhD, you will exploit proteins that contain uAAs to map key, dynamic virus-host interactions using cryo-electron tomography (cryo-ET) coupled with fluorescence and chemical observation. Cryo-ET allows us to dissect the architecture of macromolecular machinery in situ, allowing us to examine structures within the cell during the virus replication cycle. You will use this to study one the most serious viral pathogens to humans: Rift Valley Fever Virus (RVFV), a WHO priority pathogen.
This project will initially explore novel strategies for the insertion, creation and exploitation of unnatural amino acids (uAAs) based on pyrolysine (Pyl)2,3. As an example of the use of this, uAAs will allow the introduction of fluorophores into small viral proteins that are not amenable for bulky fluorescent protein labelling such as GFP. Targeted cryo-fluorescent guided focused ion beam (FIB) milling will be used to generate a lamella containing a viral target which we can image using cryo-ET at high-resolution, ultimately allowing us to place viral structures in their native cellular context. Separately, this system can facilitate the introduction of metalated uAAs into proteins to allow them to be located with high precision (4-10 nm), sufficient to find a small viral target in a large cell imaged by cryo-ET.
You will begin by applying these methods to locate and image RVFV NSs filaments inside the nucleus. Thin-section electron microscopy has identified bundles of 10 nm parallel filaments of the viral NSs protein, but no other ultrastructural details of these have been discerned and their function is unknown. Finally, our work will pursue the application of sub-tomogram averaging to the repetitive unit of the NSs filament to reach near-atomic resolution.
References: (1) L. James, mBio, 2022, 13(5). (2) C.D. Spicer, B.G. Davis, Chem. Commun, 2013, 49, 2747-2749. (3) M.K. Bilyard et al. Nature 2018, 563, 235-240.