An Analysis of Human Cytomegalovirus Gene Usage A thesis submitted in candidature for the degree of DOCTOR OF PHILOSOPHY by Sepehr Seirafian December 2012 Institute of Infection and Immunity Cardiff University Declaration This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed ………………………………………… (candidate) Date………………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of …………………………(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………… (candidate) Date ………………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loans after expiry of a bar on access previously approved by the Graduate Development Committee. Signed ………………………………………… (candidate) Date ………………………… i Acknowledgements I would like to express my sincerest gratitude to my supervisors Prof. Gavin Wilkinson and Dr Peter Tomasec for the opportunity to study a PhD, and for their constant encouragement, patience and endeavours. I wholeheartedly thank my colleagues at the Institute of Infection and Immunity for making this PhD a pleasurable experience. Special thanks to Dr James Davies and Dr Richard Stanton for their ever-present support in the laboratory, Dr Rebecca Aicheler for the NK cell inhibition assays, Dr Simone Cuff for the generation of polyclonal sera, and Dr Eddie Wang, Dr Ceri Fielding, Dr Jason Twohig and Dr Stephen Clark for sharing valuable insights. Thanks also to Daniel Sugrue for his contribution to immunofluorescence imaging, William Perks for assistance in data processing, Dr Carole Rickards for help until her retirement, and Mrs Dawn Roberts for assistance since her appointment. I wish to thank my parents for their relentless support throughout my studies at University. Special thanks must also go to my grandfather and A.Wesolowska for showing keen interest and kind concern for my progress in writing this thesis. ii Summary HCMV encodes a plethora of immune-modulating functions, many of which have yet to be assigned to specific genes. In prospect of performing high-throughput screens to identify and characterise such functions, a library of recombinant adenoviruses (RAds) each encoding a V5 epitope-tagged HCMV protein was generated. Protein expression was validated and characterised for the vast majority of RAds by western blot and immunofluorescence. HCMV has been reported to both upregulate cell surface expression of Fas, and render cells resistant to Fas-mediated killing. This thesis demonstrated that Fas levels are markedly reduced at the surface of HCMV-infected cells as an early function that persists through the late phase. Screening a panel of HCMV deletion mutants eliminated 83 genes as not required for Fas downregulation, while screening the RAd library did not identify any single HCMV gene as being sufficient for this function. Deep sequencing of the HCMV transcriptome recently led to the identification of UL150A as a novel protein-coding gene. To test this prediction, UL150A was tagged within the strain Merlin genome. UL150A was shown to encode multiple protein products, and be expressed with early and late kinetics. In a screen of the RAd library, gpUL4 was observed to be secreted from cells. To investigate this function in the context of HCMV infection, an epitope-tag was inserted at the 3’-end of the UL4 gene in the strain Merlin genome. Tagged gpUL4 was secreted from cells infected with strain Merlin. Secreted gpUL4 was more heavily glycosylated, and produced in greater abundance than its intracellular counterpart late in infection. Active secretion would be consistent with gpUL4 acting as a virokine, cytokine or cytokine/chemokine-binding protein. gpUL4 purified from supernatants of Merlin- or RAd-UL4-infected cells inhibited NK cell degranulation. Furthermore, gpUL4 did not co- purify with virus particles, indicating it is unlikely to be a virion component. iii Contents Declaration ........................................................................................... i Acknowledgements ............................................................................. ii Summary ............................................................................................ iii Contents ............................................................................................. iv List of tables ........................................................................................ x List of figures .......................................................................................xi Abbreviations .................................................................................... xiv 1-Introduction ............................................................... 1 1.1. History of isolation of HCMV .............................................................................. 2 1.2. HCMV Genome ................................................................................................... 4 1.2.1. Genome organization.............................................................................................. 4 1.2.2. HCMV Strains .......................................................................................................... 7 1.2.3. Number of genes ..................................................................................................... 9 1.2.3.1. HCMV gene usage .......................................................................................................... 9 1.2.3.2. Nomenclature of HCMV genes and proteins ............................................................... 12 1.2.3.3. Non-coding RNA molecules ......................................................................................... 12 1.2.4. Essential and non-essential genes ........................................................................ 15 1.2.5. Gene families ........................................................................................................ 18 1.3. Virion structure ................................................................................................. 18 1.3.1. Overview of structure ........................................................................................... 18 1.3.2. Nucleocapsid ......................................................................................................... 22 1.3.3. Tegument .............................................................................................................. 22 1.3.4. Envelope ................................................................................................................ 26 1.3.5. Other virion components ...................................................................................... 27 iv 1.3.6. Dense bodies and NIEPs ........................................................................................ 28 1.4. Life cycle and gene expression ......................................................................... 31 1.4.1. Tropism ................................................................................................................. 31 1.4.2. Entry ...................................................................................................................... 32 1.4.3. Productive infection/ lytic cycle ............................................................................ 33 1.4.3.1. Overview ...................................................................................................................... 33 1.4.3.2. Nuclear transport of viral DNA .................................................................................... 34 1.4.3.3. Immediate early (IE or α) genes ................................................................................... 34 1.4.3.4. Examples of Early (β) gene functions ........................................................................... 38 1.4.3.5. Late gene expression ................................................................................................... 38 1.4.3.6. Assembly and egress .................................................................................................... 39 1.4.4. Latency and reactivation ....................................................................................... 41 1.5. Prevalence, transmission, and detection ......................................................... 44 1.5.1. Prevalence ............................................................................................................. 44 1.5.2. Transmission ......................................................................................................... 45 1.5.3. Detection ............................................................................................................... 46 1.6. Clinical manifestations and treatment ............................................................. 47 1.6.1. Range of diseases .................................................................................................