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Activation of the TLR4 Signalling Complex by the Ebola Virus Glycoprotein


Type

Thesis

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Authors

Abstract

Infection by the Ebola virus, a member of the Filoviridae family of negative sense unsegmented RNA viruses, leads to acute viral haemorrhagic fever with a fatality rate of greater than 50 % and is currently incurable. As such, understanding the biochemical mechanisms and molecular interactions underpinning Ebola Virus Disease represents a clearly unmet therapeutic need. Initially, the virus replicates to very high levels in macrophages and dendritic cells, both evading recognition and also actively suppressing the innate immune system. The virus load leads to the massive release of cytokines, an unregulated cytokine storm which develops into sepsis-like haemorrhagic fever characterised by tissue damage, loss of vascular integrity and multi-organ failure. The spike glycoprotein (GP1,2), the only surface protein within the Ebola genome, has been identified as the driver for this hyperinflammatory response at the site of infection. Additionally, GP1,2 is shed into the bloodstream following proteolytic cleavage of GP1,2 by TACE protease at the virion membrane. Shed GP migrates through the bloodstream to remote tissues, activating innate immune cells leading to host-wide cytokine storms. The innate immune receptor Toll-like receptor 4, a pattern-recognition receptor (PRR), usually a bacterial LPS sensor with protective pro-inflammatory signalling, has been identified as the source of hyperinflammation during Ebola virus infection. The molecular basis of TLR4 agonism by GP1,2 and also other viral proteins is unknown to date, and this body of work goes some way to explore this interaction. This thesis explores the Ebola virus glycoprotein and the associated hyperactivation of the TLR4 signalling pathway. Understanding the role of the innate immune system in Ebola virus infection and the central role of the viral glycoprotein in the disease is vital for developing better treatment and finding potential therapeutic targets. The primary focus during this investigation was the involvement of GP glycosylation in TLR4 activation, as glycans contribute to over 50% of the molecular weight of GP and cover most of the protein surface. As TLR4 is not known to be lectin-like, capable of binding glycans, investigations into a glycoprotein of viral origin capable of TLR4 activation, could reveal novel mechanisms that may lead to therapeutic advances. To experimentally address these aims, I present the first expression of native and stable Zaire EBOV GP in HEK293 under BSL2 conditions with native folding, glycosylation, and full activity. Additionally, to allow the exploration of ideal expression conditions for native GP, multi-step protein purification strategies were developed to ensure high yields while maintaining protein integrity and avoiding contamination through LPS. The establishment of these novel methodologies laid the foundation to study the interaction between TLR4 and GP. Through establishing TLR4 activity assays I assessed the involvement of GP glycosylation through individual glycosylation mutants and processed glycans. Further, the possible interaction between the TLR4 signalling complex and GP was assessed with biochemical interaction studies. Surprisingly, although the initial hypothesis was built on the contrary, the findings suggest that the glycosylation of ZEBOV GP, does not play a specific role in the activation of TLR4 signalling complex. However, the glycosylation was found to be vital to ensure GP stability and shielding of vulnerable domains from proteases. Ultimately, I was able to set up novel EBOV GP production methods and established efficient purification strategies which may be utilised at low cost with high yield of glycosylated and stable GP. I uncovered the importance of glycosylation for GP stability, and if absent has an indirect negative impact on protein activity and consequently TLR4 activation. These discoveries and novel methodologies contribute largely towards our understanding of EBOV GP and TLR4 activation, laying the foundation for further investigations into the unusual receptor-ligand interaction and how it may be therapeutically exploited.

Description

Date

2021-10-12

Advisors

Gay, Nicholas

Keywords

Ebola Virus, Innate immunity, TLR4, Ebola Glycoprotein, Viral Haemorrhagic Fever

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Oliver Gatty Foundation