Tug-of-war: Characterising functional amyloid assemblies that trigger or terminate programmed cell death during viral infection

Abstract

Functional amyloid is an increasingly recognised type of protein assembly that can serve diverse physiological purposes. Functional amyloid is distinct from disease associated amyloid, which plays important aetiological roles in a number of irreversible and degenerative human and animal pathologies. Functional amyloid is a high molecular weight β-sheet rich protein assembly characterised by fibrillar architecture, insolubility, resistance to protease degradation and high stability. An important functional amyloid complex employed by mammals is the necrosome, which is a multi-component higher-order signalling assembly that drives the programmed cell death pathway necroptosis. Necroptosis is a crucial part of the innate immune system and is directly implicated in defense against important human pathogens. Necroptosis is mediated by protein:protein interaction sequences called RHIMs (RIP Homotypic Interaction Motifs). Due to its importance in curtailing infection, necrosome components are targeted for inhibition by pathogens using a suite of immune-evasive strategies. This thesis describes the analysis of RHIM-containing necrosome components associated with cell death signalling and virus RHIM-driven mechanisms to subvert necrosome function. The work in this thesis is divided into three major sections. In the first section, analysis of the RHIM of TRIF, an innate immune adaptor protein that responds to ligation of Toll like receptors during pathogenic infection, was performed. It is known that when activated, TRIF associates with the kinases RIPK1 and RIPK3 through RHIM-based interactions. The interactions between TRIF and these two proteins can lead to activation of downstream cell death effectors that drive necroptosis. The work in this portion of the thesis describes elucidation of the biophysical characteristics of higher-order assemblies formed by the RHIM from TRIF. TRIF is shown to spontaneously self-assemble into amyloid assemblies with unique morphology dependent on the presence of the RHIM. The residues that comprise the insoluble amyloid core of TRIF were determined by protease digestion and mass spectrometry. Single molecule confocal coincidence spectroscopy was used to demonstrate direct interactions between TRIF and RIPK3 and/or RIPK1. The size and stability of these hetero-assemblies were investigated. In the second section, a novel mechanism of inhibition of amyloid-driven cell death is described for Varicella Zoster virus, a medically important pathogenic herpesvirus. Herpesviruses can encode proteins that contain RHIM sequences. These virus RHIM sequences mediate protein:protein interactions with human RHIM-containing proteins, leading to human:virus protein hetero-assemblies that render the human proteins unable to signal for programmed cell death, which would otherwise destroy infected cells. A novel virus RHIM is described that is present in the Varicella Zoster virus protein ORF20. The RHIM from ORF20 is shown to be crucial for its infectivity. It is demonstrated that the RHIM from ORF20 is amyloidogenic, like other human and virus RHIMs. ORF20 can interact with the human cell death-associated proteins ZBP1, a nucleic acid sensor that directly responds to cytoplasmic viral ligands, and RIPK3. Interactions between ORF20 and ZBP1 result in the formation of hetero-assemblies that are larger and more stable and have morphology distinct from ZBP1 homo assemblies. These alterations in ZBP1 assembly properties are proposed to underlie the subversion of ZBP1 signalling that occurs during VZV infection. In the third section, efforts are described to use fluorescent microscopy to detect amyloid necrosomes in cell culture. Despite the growing understanding of functional amyloid in many aspects of biology, there has been limited visualisation of functional amyloid within cells by common microscopy techniques. In particular, no fluorescent microscopy of endogenous RHIM-containing proteins in their amyloid state has been reported. In this project, iterative attempts to image both the identity and amyloid structure of the RIPK1 in the necrosome were performed. These experiments were performed with amyloid staining dyes including Thioflavin T and Thioflavin S, novel amyloid-specific antibodies OC and A11, and protein-specific antibodies for the necroptosis-associated proteins RIPK1 and MLKL. Ultimately, A11 was identified as a potentially useful probe for the detection of endogenous amyloid necrosomes. Overall, the work in this thesis describes multiple aspects of functional amyloid associated with programmed cell death. Structural characterisation of cell death complexes is described in both physiological and pathological settings which are fundamental for understanding the molecular nature of innate immune signalling.