Multi-level proteomics profiling of virus-restrictive and -permissive cellular environments to discover novel regulators of virus infection

Thesis

Viruses rely on host cell resources to replicate and propagate. To mitigate, human cells can express a diverse set of antiviral proteins to combat virus infection. Understanding the host proteins that aid or repress virus infection can be instrumental for the development of novel antiviral strategies. This recent decade has witnessed rapid developments in discovering host proteins that regulate virus infection. However, our current knowledge of the landscape of host proteins that drive or restrict virus infection remains largely incomplete. Here I employed a multi-level proteomics analysis to identify novel regulators of virus infection in a model system that comprises cells highly permissive to viruses (HEK293T), cells at steady-state (HEK293), and cells restrictive to viruses (HEK293 stimulated with interferon alpha (IFN-α)). This analysis examines changes in protein and RNA abundance, phosphorylation, and RNA binding across different cellular states. Whole-cell proteomic analysis revealed that HEK293T cells are depleted of well-established antiviral factors and enriched in proteins that interact with viral protein and RNA, which could explain its high permissiveness to virus infection. Proteome-wide comparative analysis also highlights that a number of proteins exhibit distinct expression patterns across permissive, steady-state, and restrictive cellular states, including genes that have not been previously associated with IFN-α response. Further analysis comparing cellular proteome and transcriptome discloses substantial discordances between protein- and RNA-level responses after IFN-α stimulation. The analysis of RNA-bound proteome (RBPome) shows, for the first time, that IFN-a stimulation alters the protein-RNA interaction landscape in human cells. An in-depth phosphoproteomic analysis of IFN-α stimulated cells provides evidences that IFN-α could regulate the cellular RBPome through phosphorylation of RNA binding proteins. By integrating evidences from multiple levels, I have selected and generated stable expression cell lines for 19 candidate genes to test their potential regulatory roles in viral fitness. Collectively, this study provides an information-rich dataset that examines host determinants of virus-permissive or restrictive cellular states from multiple levels. This could be a valuable source for further analysis and validation studies to discover regulators of virus infection.

Authors: Chen, H

• DOI: 10.5287/ora-ko28ozndw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: proteome; interferon alpha; RNA bound proteome; transcriptome; phosphoproteome; HEK293; mass spectrometry; permissive; HEK293T; restrictive; virus infection
• Subjects: Immunology