An Exploration of the Interplay Between HSV-1 and the Non-Homologous End Joining Proteins PAXX and DNA-Pkcs

An exploration of the interplay between HSV-1 and the non-homologous end joining proteins PAXX and DNA-PKcs Benjamin James Trigg Gonville and Caius College This dissertation is submitted for the degree of Doctor of Philosophy September 2017 2. Abstract An exploration of the interplay between HSV-1 and the non-homologous end joining proteins PAXX and DNA-PKcs Benjamin James Trigg Abstract DNA damage response (DDR) pathways are essential in maintaining genomic integrity in cells, but many DDR proteins have other important functions such as in the innate immune sensing of cytoplasmic DNA. Some DDR proteins are known to be beneficial or restrictive to viral infection, but most remain uncharacterised in this respect. Non-homologous end joining (NHEJ) is a mechanism of double stranded DNA (dsDNA) repair that functions to rapidly mend broken DNA ends. The NHEJ machinery is well characterised in the context of DDR but recent studies have linked the same proteins to innate immune DNA sensing and, hence, anti-viral responses. The aim of this thesis is to further investigate the interplay between herpes simplex virus 1 (HSV-1), a dsDNA virus, and two NHEJ proteins, DNA protein kinase catalytic subunit (DNA- PKcs) and paralogue of XRCC4 and XLF (PAXX). PAXX was first described in the literature as a NHEJ protein in 2015, but whether it has any role in the regulation of virus infection has not been established. Here we show that PAXX acts as a restriction factor for HSV-1 because PAXX-/- (KO) cells produce a consistently higher titre of HSV-1 than the respective wild type (WT) cells. We hypothesised that this could be due to a role of PAXX binding viral DNA and directly inhibiting HSV-1 replication or activating an anti-viral innate immune response. We have been able to, at least partially, rule out both of these initial hypotheses by showing that there was a reduced number of viral genomes present in KO cells during active lytic infection, and that an identical level of type I interferons are produced from WT and KO cells during HSV-1 infection. Although further characterisation of HSV-1 infection in WT 3. Abstract and KO cells has not defined the molecular mechanism of restriction of HSV-1 by PAXX, we have uncovered a potential role for PAXX in mitogen-activated protein kinase (MAPK) signalling. In addition, and consistent with its function in restriction of HSV-1 infection, we show that infection with this virus in WT cells induces a loss of nuclear PAXX protein. Preliminary data suggest that these changes in localisation may occur as a result of stimulation of the cells with DNA, but not the RNA analogue poly(I:C). The role of PAXX in the regulation of HSV-1 infection in vivo was investigated by studying KO mice. Despite previous observations that mice lacking NHEJ proteins have brain defects related to autoinflammatory pathology, there were no obvious defects in the development of Paxx-/- mice, and they had brains of normal weight. No significant difference in viral spread or viral protein expression was observed between WT and KO HSV-1 infected mice, and KO mice did not exhibit abnormal pathology. There were, however, small but significant differences in the cellular immune response to infection which might be explained by reduced MAPK signalling in KO cells. DNA-PKcs is another component of the NHEJ machinery that acts to assist in dsDNA break repair in the nucleus and as an innate sensor of cytoplasmic viral DNA, but the effect of DNA-PKcs on HSV-1 infection has not been fully explored. Murine skin fibroblasts (MSFs) derived from wild type and PRKDC-/- (DNA-PKcs deficient) mice were cultured ex vivo and used for innate immune studies. Although HSV-1 was able to infect and stimulate these cells, no differences in the stimulation of innate immune gene expression between the two genotypes was observed, suggesting that DNA-PKcs does not contribute to HSV-1 sensing in MSFs. It has previously been reported that the HSV-1 protein ICP0 targets DNA-PKcs for degradation, although the reason for this is unknown. We confirmed these data, although found it to be cell-type specific and explored this interaction further using PRKDC-/- RPE-1 cells created using CRISPR/Cas9. HSV-1 infection in these cells followed unusual dynamics, and the development of cytopathic effect was accelerated as compared to WT cells. Together these observations confirm that DNA-PKcs regulates HSV-1 infection, but more work is required to fully understand the mechanisms involved. 4. Preface Preface This dissertation 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. This dissertation 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 dissertation 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. This dissertation does not exceed the prescribed word limit for the Biology Degree Committee. 5. Acknowledgements Acknowledgements First and foremost, I would like to thank Dr Ferguson for his advice and guidance over the past four years. Brian’s support has been unwavering, despite the many times that I have been an excellent test of his patience, and for this I shall always be grateful. As is often the case, aspects of this project have been a team-effort. Especial thanks go to Kathi and Paula for their help with the PAXX project, and to Zhenya for helping me to navigate the strange worlds of cellular immunity and in vivo work. Over twenty people have passed through the lab in the time that I have been doing my PhD, and each has helped make my time in the lab considerably more enjoyable. Unfortunately it is impractical to list how each has helped me, but Christian, the master of distraction, deserves a special mention for guiding me through my first, hard year. The number of people I would like to acknowledge outside of ‘The Lab of Brian’ is testament to the extent to which I have benefitted from the kindness and generosity of others. Prof. Geoffrey Smith and his lab have supported me throughout with both advice and reagents. The number of times that I bothered David Carpentier is surpassed only by his wealth of knowledge and enthusiasm to help. I have also benefitted greatly from the wisdom and charity of Helen Ewles and Yongxu Lu. The help of Heather Coleman was instrumental in facilitating many aspects of the PAXX project, and there was never a dull moment while working with her. Dan Wise provided invaluable advice on CRISPR, but was otherwise just an annoyance. Mike Hollinshead and Nigel Miller set aside a great deal of time to impart the wonders of microscopy and flow cytometry, respectively. Des Jones provided help and worldly advice in my first few years. I would also like to thank Adrian Smith and Letitia Jean for inspiring me to embark on a PhD. Without their advice and encouragement, I probably would not be writing this today. Thanks also to my long-suffering friends who have helped to preserve what is left of my sanity, be that through rowing, the pub quiz, or Mario Kart. To my even-longer- suffering family, a special mention goes to mum and Sam for their surprising eagerness 6. Acknowledgements to help with proof-reading, but thanks also to dad and Jess for all of your support and understanding over the last four years. Finally, thank you to Jo. After putting up with my unsocial hours and perpetual grumbling over the last four years she probably deserves a page to herself, but at this point in composing my thesis I’m not sure I can manage that much more writing. Suffice to say, she has provided essential moral, emotional, and nutritional support, without which I would probably not have had the stamina to see the PhD to the end. I could not have asked for a more patient and generous partner. Thank you. 7. Table of Contents Table of Conte nts Table of Contents Abstract ................................................................................................................................................... 3 Preface ..................................................................................................................................................... 5 Acknowledgements .............................................................................................................................. 6 Table of Contents ................................................................................................................................. 8 List of figures ...................................................................................................................................... 14 List of tables ........................................................................................................................................ 16 Abbreviations ...................................................................................................................................... 18 1 Introduction ............................................................................................................................... 22 1.1 The DNA Damage

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