The Role of RL13 in Human Cytomegalovirus Pathogenesis A thesis submitted in candidature for the degree of DOCTOR OF PHILOSOPHY (PhD) by Carmen Grace Bedford December 2018 Division of Infection and Immunity, School of Medicine, Cardiff University Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ……………………………………………………… Date ………………….…………….……… STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed ………………………………………….…………… Date …………………………….…………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third-Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ……………………………………….……….…… Date …………………….………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed …………………………………………….…..….. Date ………………………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ……………………………………………..……… Date ………………………………….……… I II Acknowledgements I would first like to express my sincerest gratitude to my supervisors for the opportunity to undertake this PhD project. In particular to Dr Richard Stanton, whose support, guidance and patience with me throughout made this possible. Thank you for challenging me and allowing me to develop professionally and personally. I am always amazed at your capacity to multitask and your ability to appraise and return a piece of work so quickly! I am also very grateful to Prof Gavin Wilkinson for his guidance and valuable insight and to Dr Peter Tomasec who sadly is no longer with us but is fondly remembered. I would like to thank Health and Care Research Wales who have funded this thesis. I am very appreciative to Prof Robin Antrobus, Dr Mike Weekes and Dr Paul Lehner who performed the mass-spectrometry analysis of virions used in this work. I am indebted to all members of the HCMV and Adenovirus Research groups for their friendship, assistance and encouragement without which I would have struggled. Thank you, Dawn Roberts, for your help and answering my “stupid questions”, Dr Evelina Statkute for your guidance and Dr Isa Murrell for your constant enthusiasm in what RL13 does. Thank you, Dr Mihil Patel, for your company during Bake-Off season, Dr Simone Forbes for all the caffeine-fuelled laughs, and Dr Hester Nichols for your continued friendship and for maintaining my sanity with endless cups of tea and a great taste in films! Thank you to my parents, Mike and Linda, and the rest of the Bedford clan for their love, kindness, generosity and constant support. Tori, thank you for all the weekends and evenings away from the lab winning pots in the double – to this day no one knew who they were or what they were doing. Finally, to my fiancé Nathan, thank you for believing in me even when I didn’t believe in myself. Without you keeping me together I would never have made it here. I look forward to our next adventure together. “You miss 100% of the shots you don’t take. - Wayne Gretzky” - Michael Scott III IV Summary Human cytomegalovirus (HCMV) is the leading infectious cause of congenital malformation. In vitro propagation of HCMV strains that authentically represent clinical virus are required to facilitate effective research. However, this is problematic, since efficient replication of clinical HCMV strains in vitro is achieved only after mutations have been selected. The first gene to mutate is consistently RL13. RL13 encodes a virion envelope protein that suppresses release of infectious virus from cells. Despite mutation in cell culture it is conserved in clinical strains, indicating that it is important in vivo. However, it is unclear how RL13 inhibits virus in vitro, or what advantage it provides in vivo. RL13 expression did not affect genome replication or virion protein production, however it reduced the infectivity of released virions. Mass spectrometry revealed that RL13 expression altered the composition of released virions, however there was no obvious reason for these changes to effect infectivity. During virus spread, RL13 promoted cell- cell transfer of virus, reducing the ability of neutralising antibodies to inhibit virus spread in fibroblasts, however this effect was masked by UL128L expression in other cell types. Genetic analysis of RL13 from clinical strains revealed that RL13 sequences clustered into 9 distinct clades, with sequences from different clades retaining the ability to inhibit the release of infectious virus. A RL13 homologue from Rhesus CMV also restricted the release of infectious virus. This conservation of function suggests that the phenotype is not an artefact of in vitro culture conditions. RL13 on the cell surface bound human IgG, however it did not inhibit antibody-dependent cellular cytotoxicity during infection. This thesis has delivered insights into the role of RL13 during infection, has demonstrated the importance of using more clinically relevant RL13+ HCMV strains and will aid future research using such HCMV strains. V VI Table of Contents Declaration ...................................................................................................................................... I Acknowledgements ....................................................................................................................... III Summary ......................................................................................................................................... V Table of Contents ......................................................................................................................... VII List of Figures ................................................................................................................................ XII List of Tables ................................................................................................................................ XIV CHAPTER 1. INTRODUCTION ........................................................................................................ 1 1.1 Human Cytomegalovirus (HCMV) ......................................................................................... 2 1.1.1 The Herpesvirus Family ................................................................................................ 2 1.1.2 Discovery and Isolation ................................................................................................ 4 1.2 Clinical Importance and manifestations of HCMV infection ................................................ 5 1.2.1 Epidemiology ................................................................................................................ 5 1.2.2 Transmission and Primary Infection ............................................................................. 5 1.2.3 Infection in the Immunocompetent Host ..................................................................... 6 1.2.4 Congenital and Perinatal Infection ............................................................................... 6 1.2.5 Infection in at-risk groups ............................................................................................. 7 1.2.6 Treatments and Vaccination ........................................................................................ 8 1.3 Viral Particles ...................................................................................................................... 10 1.3.1 Components of Infectious virions ............................................................................... 10 1.3.2 Non-infectious Viral Particles ..................................................................................... 12 1.4 Viral Lifecycle ...................................................................................................................... 16 1.4.1 Virus Entry .................................................................................................................. 16 1.4.2 Viral DNA Replication and Gene Expression Regulation during Infection .................. 16 1.4.3 Viral Particle Maturation and Egress .......................................................................... 20 1.4.4 Latency and Reactivation ........................................................................................... 23 1.5 HCMV Infection .................................................................................................................. 24 VII 1.5.1 Intra-host Dissemination and Tropism ...................................................................... 24 1.5.2 Lessons from HCMV Infection in vitro ....................................................................... 25 1.5.3 The Immune Response and Immunomodulation ...................................................... 26 1.6 The Genetics of HCMV ......................................................................................................