Identification and Characterisation of Human Cytomegalovirus-Mediated Degradation of Helicase-Like Transcription Factor Kai-Min Lin Department of Medicine Cambridge Institute for Medical Research University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy Fitzwilliam College September 2020 Declaration I hereby declare, that except where specific reference is made to the work of others, the contents of this dissertation are original and have not been submitted in whole or in part for consideration for any other degree of qualification in this, or any other university. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as where specified in the text and acknowledgments. This dissertation does not exceed the specified word limit of 60,000 words as defined by the Degree Committee, excluding figures, photographs, tables, appendices and bibliography. Kai-Min Lin September, 2020 I Summary Identification and characterisation of human cytomegalovirus-mediated degradation of helicase-like transcription factor Kai-Min Lin Viruses are known to degrade host factors that are important in innate antiviral immunity in order to infect successfully. To systematically identify host proteins targeted for early degradation by human cytomegalovirus (HCMV), the lab developed orthogonal screens using high resolution multiplexed mass spectrometry. Taking advantage of broad and selective proteasome and lysosome inhibitors, proteasomal degradation was found to be heavily exploited by HCMV. Several known antiviral restriction factors, including components of cellular promyelocytic leukemia (PML) were enriched in a shortlist of proteasomally degraded proteins during infection. A particularly robust novel ‘hit’ was helicase-like transcription factor (HLTF), a DNA repair protein that participates in error-free repair of stalled replication forks. HLTF was found degraded very early during infection, and its expression remained low throughout the course of HCMV lytic cycle. De novo expression of UL145, a previously uncharacterized viral protein, was found necessary and sufficient to degrade HLTF via recruitment of the cullin 4/DDB1 E3 ligase complex. HLTF degradation was reported in human immunodeficiency virus (HIV) infection, however the interaction between HLTF and viruses remain largely elusive. The roles of UL145 were explored in hopes of understanding functions of HLTF in HCMV infection. UL145 was identified as a non-essential immediate early protein, however had a possible role in type I interferon (IFN) induction regulation in later stages of HCMV lytic progression. As the key host protein to be rescued by UL145 deletion, depletion of HLTF was found to transiently impair IFNβ transcription and HCMV infection. I hypothesise that HLTF is an undiscovered nuclear viral DNA sensor that triggers an antiviral interferon response during viral DNA replication. Additionally, work presented here expands the range of powerful screening technologies to identify HCMV restriction factor candidates by identifying virally degraded host proteins. Further investigation of these candidates will contribute to our II understanding of how HCMV modulates host protein expression to evade antiviral factors. III Acknowledgments Firstly, I would like to express my sincere gratitude to my supervisor Dr. Mike Weekes, for his everlasting support, guidance, and encouragement throughout the pursuit of this degree. I was blessed with this opportunity for he patiently reviewed a PhD application coming abruptly. His instruction was fundamental to this thesis and helped me develop as a research scientist. Without his scientific acumen and aweing diligence, it was nearly impossible to move this challenging project forward. I would also like to thank everyone in the Weekes lab, who created the most enjoyable working environment in the world that I was almost reluctant to leave. Many of them contributed a lot to this project, especially Dr. Katie Nightingale, who laid a firm foundation for the entire protein degradation project and generously provided helpful discussions. I really appreciate Dr. Luis Nobre, Alice Fletcher-Etherington, and Dr. Ben Ravenhill for providing me the many cell lines and plasmids I needed for my experiments, and Dr. Lior Soday for the help on processing the proteomics data. All members of the Weekes lab were truly the highlights of my PhD. I will deeply miss the wise decisions from Katie, reliable solutions from Luis, warm support from Lior, friendly chats with Colin, evening enlightenments from Ben, prodigious work from Alice, wee comedies from Leah, insider gossips from Cassie, and of course, having Martin to check on my wellbeing. Our collaborators at Cardiff University, particularly Dr. Rich Stanton, had unfailingly supplied us with all kinds of HCMV strains. His expertise in HCMV provided reliable protocols and useful advice that helped overcome many hurdles. My research involved many equipment from CIMR core facility. I would like to thank Dr. Robin Antrobus from the proteomics core, Dr. Reiner Schulte from the flow cytometry core, Mark Bowen from the Microscopy core, and Mark Smallwood the level coordinator for maintaining the state of the art facilities and the help making the experimental plans into reality. I am very fortunate to receive this opportunity to study at the University of Cambridge as an oversea fee status student. I would like to thank the Cambridge Commonwealth, European & International Trust and the Ministry of Education of Taiwan for setting up IV and running the Taiwan-Cambridge scholarship programme, which funded my tuition, fee, and stipend in Cambridge. Finally, I will not be able to complete this without the unconditional support I have received from my friends and family, no matter from Cambridge, the EBV group of National Taiwan University, National Tsing-Hua University, or anywhere else I have ever called home. I would like to thank Julia Woitischek and Ragini Medhi for accompanying me on this PhD journey. I am also very grateful to Chin-Wen Liang and Chung-Ying Huang for bearing with me during all the ups and downs tirelessly in the past decade. V VI Table of Contents Declaration……….…………………………………………….………………………………..……………………………….I Summary………………………………………..……………………………….………………………………………II Acknowledgement…………………………………………………………….……….…………………….….…IV Table of Contents……………………………………………………………….……………………………….…VII List of Figures……………………………………………………………………….……………………………..…XII List of Tables………………………………………………………………………………………………..……….XVI Abbreviation....................................................................................................…..XVIII Chapter 1. Introduction ......................................................................................... 1 1.1. Human Cytomegalovirus .................................................................................... 1 1.1.1. Herpesvirdae family .................................................................................... 1 1.1.2. Human Cytomegalovirus ............................................................................ 2 1.1.3. Virion structure ........................................................................................... 3 1.1.4. Viral genome ............................................................................................... 4 1.1.5. Laboratory strains ....................................................................................... 5 1.1.6. Latency ........................................................................................................ 6 1.1.7. Lytic replication cycle ................................................................................. 7 1.1.8. HCMV-associated diseases ....................................................................... 11 1.1.9. Intervention .............................................................................................. 11 1.2. Viral DNA Sensors for Herpesviruses and Viral Counteractions ...................... 12 1.2.1. Interferon.................................................................................................. 12 1.2.2. Type I interferon production and signalling ............................................. 14 1.3. Protein degradation in the cell ........................................................................ 21 1.3.1. Ubiquitin-proteasome pathway ............................................................... 21 1.3.2. Viral interference of proteasome-mediated degradation ....................... 26 1.3.3. Lysosomal proteolysis ............................................................................... 27 1.3.4. Viral subversion of lysosomal degradation .............................................. 31 1.4. DNA repair........................................................................................................ 34 1.4.1. DNA repair and herpesviruses .................................................................. 35 1.4.2. Helicase-like transcription factor (HLTF) .................................................. 37 VII 1.5. Multiplexed proteomic analysis screening of host protein degradation during early HCMV infection .................................................................................................. 39 1.5.1. Protein with E3 ligase domain was targeted for degradation during HCMV infection .................................................................................................................