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Proteomic Investigation of Interferon Alpha Stimulation and Viral Regulation to Identify Candidate Antiviral Restriction Factors

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Proteomic Investigation of Interferon Alpha Stimulation and Viral Regulation to Identify Candidate Antiviral Restriction Factors Proteomic Investigation of Interferon Alpha Stimulation and Viral Regulation to Identify Candidate Antiviral Restriction Factors Lior Vered Soday Department of Medicine Cambridge Institute for Medical Research Jesus College This thesis is submitted for the degree of Doctor of Philosophy University of Cambridge May 2020 i DECLARATION This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my thesis has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit for the relevant Degree Committee. Lior Vered Soday May 2020 ii Proteomic Investigation of Interferon Alpha Stimulation and Viral Regulation to Identify Candidate Antiviral Restriction Factors Lior Vered Soday SUMMARY 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 iii 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. iv ACKNOWLEDGEMENTS There are many individuals I would like to thank for enabling me to conduct this research, and for making my PhD experience so enjoyable. First and foremost, I would like to thank my supervisor, Dr Mike Weekes, for his advice, guidance and support throughout the PhD. He has been instrumental in my development as a researcher, and I am extremely grateful for the opportunity to perform this research in his laboratory. I would also like to thank all the members of the Weekes lab. The friendly atmosphere and everyone’s willingness to help has made it a great place to work. In particular, I would like to thank Dr Katie Nightingale, Dr Luis Nobre and Alice Fletcher-Etherington for providing me with many of the cell lines used in this research, and Dr Colin Davies for fractionating my samples. Aside from scientific help, I would also like to thank them for creating such a lovely environment in the lab. They have kept me smiling throughout the joys and the struggles of the PhD, and I am so fortunate to have spent my time in the lab with them. There are a number of others I would like to acknowledge who have also contributed to this work. I would like to thank Dr Robin Antrobus, from the CIMR Proteomics Core Facility, and Dr James Williamson from the Lehner Lab, who ran the proteomic samples discussed in this thesis on the mass spectrometer. The members of the Smith Lab, in particular Dr Jonas Albarnaz and Dr Yongxu Lu, who enabled all the research on vaccinia virus presented here. The vaccinia project has been both interesting and enjoyable, and I am extremely grateful for the opportunities the collaboration has provided. Publishing this work as a joint first author, and presenting it at the poxvirus conference in Taiwan, were highlights of my PhD. I would also like to thank the Wellcome Trust for the funding that made all this possible. I would not be here without the help and encouragement I have received throughout my studies; from teachers at The Latymer School, to supervisors at Newnham, and all the scientists I have been so fortunate to work with during summer research projects, v undergraduate projects, and lab rotations. I am so grateful for everyone that has taught and guided me. Finally, thank you to my friends and family. The Newnham girls, who have been a massive part of what first made me love being in Cambridge. Tori and Krittika for being here throughout the ups and downs of the last four years, and always with cheese. El for making Marshall Road a true home. Life in Cambridge would not have been the same without you all. Thank you to Rajiev, Chadwell Heath is not where I imagined I would be writing the last of this thesis, but there is no where I would rather have spent the COVID lockdown. I am so glad I get to finish ‘our’ PhD here with you. Thank you to all my family, Booba and Zada, those in Israel, and especially to Alan and Adi for always being there. Finally, to Mum. There are not the words to say how grateful I am for everything you do. Anything I achieve is because of the opportunities you have given me, and your encouragement, love and support. Thank you. vi PUBLICATIONS Key manuscript referenced in thesis Soday L, Lu Y, Albarnaz JD, Davies CTR, Antrobus R, Smith GL, Weekes MP. “Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist”. Cell Reports (2019) 27: 1920-1933.e7. Other articles published during PhD Ravenhill BJ, Soday L, Houghton J, Antrobus R, Weekes MP. “Comprehensive cell surface proteomics defines markers of classical, intermediate and non-classical monocytes”. Sci Rep (2020) 10: 1–11. Nobre L, Nightingale K, Ravenhill BJ, Antrobus R, Soday L, Nichols J, Davies J, Seirafian S, Wang ECY, Davison AJ, Wilkinson GWG, Stanton RJ, Huttlin EL & Weekes MP. “Human cytomegalovirus interactome analysis identifies degradation hubs, domain associations and viral protein functions”. Elife (2019), 8: 1–35 Nightingale K, Lin KM, Ravenhill BJ, Davies C, Nobre L, Fielding CA, Ruckova E, Fletcher-Etherington A, Soday L, Nichols H, Sugrue D, Wang ECYY, Moreno P, Umrania Y, Huttlin EL, Antrobus R, Davison AJ, Wilkinson GWGG, Stanton RJ, Tomasec P, Weekes MP. “High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms”. Cell Host Microbe (2018), 24: 447-460 e11 Ersing I, Nobre L, Wang LW, Soday L, Ma Y, Paulo JA, Narita Y, Ashbaugh CW, Jiang C, Grayson NE, Kieff E, Gygi SP, Weekes MP & Gewurz BE. “A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells”. Cell Rep (2017), 19: 1479– 1493 TABLE OF CONTENTS vii TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................. vii LIST OF FIGURES ...................................................................................................... xii LIST OF TABLES ........................................................................................................ xv ABBREVIATIONS ..................................................................................................... xvii CHAPTER 1: INTRODUCTION ................................................................................. 1 1.1 Introduction ............................................................................................................
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