This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. i Functional Analysis of Ovine Herpesvirus 2 Encoded microRNAs. Aayesha Riaz Thesis presented for the degree of Doctor of Philosophy The University of Edinburgh 2014 i ii Declaration I hereby declare that this thesis is of my own composition, and that it contains no material previously submitted for the award of any other degree. The work reported in this thesis has been executed by myself, except where due acknowledgement is made in the text. Aayesha Riaz 2014 Division of Infection and Immunity The Roslin Institute and R(D)SVS University of Edinburgh Easter Bush Edinburgh EH25 9RG ii Acknowledgements I am thankful to Almighty Allah, most Gracious, who in His infinite mercy has guided me to complete this PhD work. May Peace and Blessings of Allah be upon His Prophet Muhammad (peace be upon him). I want to pay deep felt gratitude to my supervisors Dr. Bob Dalziel and Prof. John Hopkins for their continuous and sustained guidance, mentoring and supervision. Truly, it was an honour and a matter of great pride to work with such dedicated people. It was Dr. Dalziel who found me raw and set the tone for this long and arduous journey. He helped me through the tough initial phases of this lengthy journey. He understood my limitations and was always there to extend a helping hand whenever I needed it. I can vividly recall those moments of anguish and desperation when my project hit difficulties and he would come up with innovative ideas or guide me towards other professionals who could help me out. He also appreciated my family constraints and also those related to my work when I was not able to be present in Edinburgh. Prof. Hopkins was never less helpful. His always open door and reassuring attitude kept my spirits high to reach this goal. I never found him to be less than encouraging and he never lost patience with my stutters. I am also appreciative to Dr. Finn Grey who never raised an eyebrow when I approached him to discuss intriguing and complex issues that either related to the lab work and / or any other technical matters related to my project. iii Dr. George Russell, Dr. Amy Buck and Mrs. Liz Thornton were kind enough to give cells and psi-M23-2 construct for use in this study. I would also like to thank Mr. Mick Watson, Miss Alison Downing and Miss Frances Turner for helping me carry out the bioinformatics analysis. I would also like to thank Dr. Bernadette Dutia and Mrs. Liz Archibald for their kind help and support whenever I needed. I was so lucky to find the other members of Dalziel group; Claire Levy, Pete Wasson, Suzanne Esper, Katie Nightingale and particularly Inga Dry who worked with me not only as colleagues but as friends. Also, I would like to thank the members of Finn's group; Natalie Reynolds, Jon Pavelin and particularly Stephen Chiwashe. They worked with me for long hours to ease me through many complex situations. Special thanks to Shoko for both moral and physical support, particularly when she was so kind to share her flat with me when I needed it the most. I also consider myself lucky to have friends like Gigi, Shuo, Xuan, Yoshi, Meng Meng and Aya. The laughs and moments of sorrow that we shared together were so unforgettable. I am also thankful to Ian, Anton, Yvonne, Marlynne, Karen, Dung, and Amr for their time to time support in and out of the lab. I would also like to thank the Higher Education Commission of Pakistan for giving me the opportunity to come to the University of Edinburgh for PhD and for funding my work. Last but not the least I am thankful to my family, especially my husband Zubair for his endless support, my kids Abdullah and Tehreem and my parents for their continued support and unending prayers. iv Abstract Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post- transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that v out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species. vi LAY SUMMARY Malignant catarrhal fever (MCF) is a fatal disease of cattle and deer caused by infection with the virus ovine herpesvirus 2 (OvHV-2). Sheep are infected soon after birth, carry the infection for life but never show signs of any disease. If cattle catch this virus from sheep they develop MCF and usually die. This disease is economically important not only in the UK but also in Sub-Saharan Africa and other areas of the developing world where it places a major burden on food production. In sheep the virus infects, and then lies dormant for the life of the animal, in cells of the immune system, these cells continue to function normally. In cattle the same type of cell is infected but they change such that they now become aggressive and attack and kill other cells in the body, resulting in disease and death. A major question in understanding how OvHV-2 causes disease is: why, given that sheep and cattle are closely related species, do sheep survive and cattle die? Herpesviruses normally only infect one species and have co-existed and evolved with that species for millions of years. A previous study in the group identified molecules, termed microRNAs (miRNAs), produced by the virus that have the potential to control the way the infected immune cell functions. miRNAs work by affecting how much of a particular protein is produced in a cell and they do this by binding very specifically with the mRNA (“blueprint”) for this protein and targeting it for destruction. The hypothesis is that the virus miRNAs have evolved to work in sheep immune cells and allow the virus to colonise these cells and survive long term. In order to investigate this hypothesis it is necessary to identify the viral and cellular proteins targeted by these miRNAs, this formed the basis of this thesis. Herpesviruses can either grow in a cell and produce new virus or remain dormant for long periods of time. I first showed that virus miRNAs can affect the production of three virus proteins , important in controlling whether the virus is dormant or growing. The control of this step may differ in sheep in cattle and may influence disease. I then investigated which cellular mRNAs (and so proteins) are targeted by virus miRNAs in sheep and cattle. I used a novel method which allows the purification of miRNAs bound to their target mRNAs. The mRNAs are then analysed and the protein they produce identified. This complex technique identified a number of cellular mRNAs (proteins) which are potentially targeted by the virus miRNAs.