Structural studies of prototype foamy virus by electron cryomicroscopy
File(s)
Author(s)
Calcraft, Thomas
Type
Thesis
Abstract
Foamy Viruses or Spumaretrovirinae are an unusual subfamily of the Retroviridae, being the most ancient extant retrovirus family as well as seemingly nonpathogenic. Prototype Foamy Virus is the FV which can infect humans. Due to its nonpathogenicity and broad tropism, PFV-based vectors for gene therapy have the potential to integrate transgenes into a broad range of host cells in vivo, with promising safety and efficacy shown in early experiments. As in the other retroviral genera, FV virions are primarily composed of Gag and Env proteins. Gag and Env show poor sequence conservation yet maintain conserved features of 3D structure, which can be compared between retroviruses to establish structural phylogenies. Structural studies of FV Gag and Env proteins will therefore shed light on both the evolution of retroviruses and the structural bases of FV infection which underpin their suitability as gene therapy vectors. I aimed to characterise PFV structurally in intact virus particles, preserving the native context of the membrane-embedded Env glycoproteins and the assembled multimeric Gag cores. By making use of electron cryomicroscopy (cryoEM), I characterise whole virus particles by electron cryotomography (cryoET) and subtomogram averaging, visualising the molecular architecture of internal core assemblies and external Env lattices. I also carry out cryoEM single particle analysis to solve atomic structures of Env and the Gag CA domain within intact virus particles. This represents the first time that a retroviral membrane glycoprotein has been resolved to de novo model-building resolution (<4Å) in situ with its full transmembrane region and membrane-proximal external region (MPER), and the first structure determination to such resolution of an assembled retroviral capsid within intact virus particles. The structures solved here offer insights into retroviral evolution, including unexpected structural homologies with viruses outside the retrovirus family, and the Env structure will benefit the effort to develop PFV-based gene therapy vectors.
Version
Open Access
Date Issued
2022-11-18
Date Awarded
01/04/2023
License URL
Advisor
Rosenthal, Peter
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)