PROBING PROTEIN-PROTEIN INTERACTIONS FROM HUMAN RESPIRATORY SYNCYTIAL VIRUS.

Abstract

Human respiratory syncytial virus (hRSV) is the leading cause of viral respiratory tract infections in children.1 In 2013 there were over 60 000 infants hospitalised in the USA alone and the virus resulted in approximately 2.1 million outpatient visits by children under 5 years old.2 The virus leaves the lungs weakened and open to further secondary infections such as bronchitis and pneumonia, especially in immunocompromised and elderly patients. Infections can lead to long-term effects and up to 70% of infants are left with respiratory problems for up to 10 years following hRSV bronchiolitis.3 For otherwise healthy children the treatment is usually supportive, using hydration and additional oxygen.1 Previous antiviral agents and decongestants have been used to little positive effect, leading to the need for new antiviral agents.
In the work described in this thesis, key protein targets have been identified from the 11 protein hRSV Genomes. It has been found that F, M2-1, M and N are integral for structural roles, transcription and virus maturation.4 M2-1 protein is a transcription factor and has long been a target for structural biologists due to its integral role in the virus. Chapter 2 describes the characterisation of the protein using biophysical techniques to obtain data on its stability and overall fold. Further work to crystallise the protein is also included.5
Chapter 3 builds on this work introducing the interaction between matrix protein and M2-1. The interaction is characterised through Surface Plasmon Resonance (SPR) and Fluorescence Anisotropy (FA), which gave equilibrium constants and thermodynamic pathways for the first time.
Chapter 4 introduces the interaction between hRSV matrix protein and hRSV fusion protein. Previous work has characterised the fusion protein and identified that the cytoplasmic tail is the key subunit which binds to matrix protein.6 Through mutagenesis of a single residue, F572 was identified as the key binding residue.7 The terminal six amino acids were used to form a peptide which formed the basis for the experiments in this thesis. The work described here presents in vitro data which will build on the published literature to further understand the interaction. SPR and FA provide experimental evidence for a hypothetical binding model. This binding model and the resulting equilibrium constants are discussed and evaluated.

The X-ray crystal structure of hRSV N protein is known8,9 and this information can be used to design ligands with the aim of disrupting the protein’s function. The work outlined here aimed to evaluate the binding of a series of ligands that were identified by Arrow Therapeutics Ltd as having antiviral activity.10 Thermal shift assays were used to understand the stabilisation of the protein in the presence of the ligand. Dynamic light scattering showed the formation of aggregates of protein when exposed to various concentrations of ligand. Cryo-electron microscopy shows association of the ligand to the protein and the consequent aggregates which form. This provided an indication of how the ligands could interact with the protein and suggests possible modes of action.
In Chapter 6, hRSV M protein’s interaction with hRSV F was further investigated. The interaction is key for the maturation of the virus11 and if the binding could be diminished, the infectivity of the virus would suffer. A pocket at the dimer interface was identified, which opened up an opportunity for computational docking studies. Through the application of biophysical techniques and computational methods,12 novel ligands were identified and the initial characterisation of their effect on protein stability was probed.
Key protein interactions which are involved in the maturation of the virus and its fusion to the host cell have been characterised and evaluated in this thesis. The opening chapters begin within basic protein characterisation and build to show protein interactions in vitro. In the later chapters, ligand subsets are identified with the aim of disrupting protein interactions or the protein folding. Characterisation of the protein ligand interactions showed protein changes and destabilisation, leading the way for further studies.