Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.814551
Title: Proteomic investigation of interferon alpha stimulation and viral regulation to identify candidate antiviral restriction factors
Author: Soday, Lior
ISNI:       0000 0004 9354 2246
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Abstract:
Antiviral restriction factors (ARFs) are host proteins that play key roles in inhibiting viral infection. The plasma membrane provides a critical interface between the cell and the virus, meaning that proteins present here are well situated to act as ARFs. An understating of these ARFs and how viruses interact with them, is crucial to our knowledge of infection and immunity, and provides potential therapeutic targets. In this thesis, I apply quantitative, multiplexed proteomic technologies to explore two characteristic features of ARFs: induction by interferon (IFN), and downregulation by viral infection. Proteomics was used to investigate cell surface changes in primary monocytes and CD4+ T-cells upon stimulation with IFNα, across five donors. The cell surface proteomes were characterised, and found to be remarkably invariant between donors, whilst the effects of IFN were more variable between donors. IFN stimulation of several proteins was validated by flow cytometry, and TMEM123 was identified as the only protein, aside from MHC class I molecules and the known restriction factor Tetherin, to be consistently upregulated by IFN at the cell surface in all donors for both cell types. Additional proteomic screens investigated the effects of vaccinia virus (VACV) infection, quantifying ~9000 human proteins over a single replication cycle. This revealed downregulation of 265 proteins including innate immune proteins, collagens and cadherins. Overlap with a previous proteomic investigation of human cytomegalovirus (HCMV) infection demonstrated that many classes of proteins were commonly targeted, suggesting an important role in infection and immunity. Of the proteins downregulated by VACV, ~70 % were proteasomally degraded. In addition to human proteins, ~80 % of all VACV proteins were quantified. These were classified into four distinct temporal classes, which correlated well with previous literature, and with known protein functions. Host-virus interactions were investigated by matching temporal profiles of human and viral proteins. HDAC5 was found to be targeted for proteasomal degradation by the viral protein C6. Overexpression and knockdown of HDAC5 demonstrated its restriction of both VACV and herpes simplex virus-1 replication. Finally, both datasets were considered alongside published proteomic data on other viral infections, in particular HCMV and HIV, to identify putative ARFs. Expression of endothelin converting enzyme 1 (ECE1) was stimulated by IFN at the surface of CD4+ T-cells and was downregulated during HCMV infection. Follow-up studies to determine if ECE1 restricts HCMV have thus far led to inconclusive results. Overall, these investigations into IFN stimulation and VACV infection have yielded very useful data, and provide valuable, comprehensive resources for future research.
Supervisor: Weekes, Michael Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.814551  DOI:
Keywords: proteomics ; innate immunity ; virology ; vaccinia ; HCMV ; interferon ; systems biology ; host-pathogen interaction ; antiviral restriction factor
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