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Characterising the Role of the Cajal Body During a Productive Adenovirus Infection

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Characterising the Role of the Cajal Body During a Productive Adenovirus Infection Characterising the role of the Cajal Body during a productive adenovirus infection Laura White Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences December 2012 The candidate confirms that the work submitted is their own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied in the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2012, The University of Leeds, Laura White i Acknowledgements I would firstly like to say a massive thank you to my supervisor, Professor Eric Blair, for his excellent guidance and support, for allowing me the freedom to direct my own research and for keeping my health in check with a constant supply of strawberries. I would also like to thank Dr Andrew Macdonald and Professor Adrian Whitehouse for their constructive critique of my work over the last three years. I am indebted to Gareth Howell for the flow cytometry and confocal microscopy assistance and to Dr Joan Boyes and Rosie Doble for their help with qPCR. I would also like to acknowledge past and present members of the lab for their help and support and for making the lab a really enjoyable place to work; Kathryn Hall, Natalie Fox, Oliver Watherston, David Orchard-Webb, Rebecca Caygill, Paul Bowles, James Findlay, Rosie Doble, Adam Lee and Irene Bassano. Many thanks go to Hazel Fermor for her generous hostessing and to Sian Tanner for charitably agreeing to live with me and for introducing me to the cultural delights of ballet and opera. I am hugely grateful to Elizabeth Glennon, Nicola Ooi, Helen Beeston, Rupesh Paudyal and James Lloyd for the weekly lunches, regular pub trips, random excursions and for generally keeping me sane. I am indebted to Bethan Thomas for patiently listening to me moan about science and for feeding me for most of the past year. Finally, I would like to give a huge thank you to my family, for their unwavering support and for always pretending that having me visit was a pleasure, no matter how grumpy and stressed I was. ii Abstract Human adenoviruses (Ads) are DNA viruses believed to hold potential as gene therapy vectors. However, Ad vectors often express residual late structural proteins, which stimulate immune responses resulting in rapid clearance of the vector. Therefore a greater understanding of the mechanisms controlling Ad late gene expression is required. During the late phase of adenovirus 5 (Ad5) infection it was previously shown that a nuclear compartment involved in RNA metabolism known as the Cajal body (CB) is disassembled from 1-6 punctate domains per cell into numerous microfoci. Furthermore, the marker protein of CBs, p80 coilin, was suggested to play a role in the expression of Ad late phase proteins. However, the exact function of coilin in Ad late protein expression is unknown. The aim of this investigation was to determine the roles of coilin and additional CB proteins during Ad infection. Immunofluorescence microscopy was utilised to investigate the redistribution of key CB proteins following Ad5 infection of A549 cells. Whilst coilin was redistributed from CBs into microfoci, another CB protein, termed survival of motor neuron (SMN), was redistributed from CBs into the nucleoplasm. To assess the roles of coilin and SMN during Ad5 infection, coilin was depleted in A549 cells by RNA interference (RNAi). Cells were infected with Ad5 and the virus yield, Ad proteins levels and Ad mRNA levels were assessed. Depletion of coilin reduced the virus yield and decreased the synthesis of Ad early, intermediate and late phase proteins. Although Ad mRNA expression was mostly unaffected, nuclear export of Ad mRNAs was abrogated in coilin-depleted cells. This indicated that coilin plays a role in Ad mRNA transport. Similar to coilin, SMN depletion significantly reduced the virus yield. However, in contrast to coilin, SMN depletion resulted in significant decreases in the levels of Ad capsid proteins, whilst non-structural proteins were either increased or unaffected. SMN depletion was found to reduce early gene transcription and altered alternative splicing patterns of Ad mRNAs. This suggested that SMN plays a role in Ad transcription and mRNA splicing. This investigation uncovered the involvement of two CB proteins in two very distinct roles during Ad infection. This is the first report suggesting a role for CBs in mRNA export. Further study is now required to identify the exact function of coilin in Ad mRNA export. Furthermore, investigation of a role for coilin in cellular mRNA export and mRNA export during infection with other viruses is also warranted. Although SMN was known to play a role in cellular mRNA splicing, this is the first report indicating that SMN may be involved in splicing of virus mRNAs. Additional work is required to define the precise function of SMN in transcription and mRNA splicing during Ad infection and during infection with other virus species. iii Table of Contents Acknowledgements ....................................................................................................................... ii Abstract ........................................................................................................................................ iii Contents ........................................................................................................................................ iv List of Figures ................................................................................................................................ x List of Tables .............................................................................................................................. xiv Abbreviations ............................................................................................................................... xv 1. Chapter 1 - Introduction ......................................................................................................... 1 1.1 Introduction ....................................................................................................................2 1.2 Adenoviruses ..................................................................................................................4 1.2.1 Adenovirus classification .......................................................................................4 1.2.2 Pathology and disease associated with human adenovirus infection .....................4 1.2.3 Adenoviruses as gene therapy and oncolytic vectors .............................................5 1.2.4 Adenovirus structure ..............................................................................................8 1.2.1.1 Major capsid components ................................................................................10 1.2.1.1.1 Hexon (polypeptide II) ...............................................................................10 1.2.1.1.2 Penton base (polypeptide III) .....................................................................10 1.2.1.1.3 Fibre (polypeptide IV) ...............................................................................10 1.2.1.2 Minor capsid components ................................................................................11 1.2.1.2.1 Polypeptide IIIa ..........................................................................................11 1.2.1.2.2 Polypeptide VI ...........................................................................................11 1.2.1.2.3 Polypeptide VIII .........................................................................................11 1.2.1.2.4 Polypeptide IX ...........................................................................................12 1.2.1.3 Core proteins ....................................................................................................12 1.2.1.3.1 Polypeptide V .............................................................................................12 1.2.1.3.2 Polypeptide VII ..........................................................................................12 1.2.1.3.3 Mu (polypeptide X) ....................................................................................13 1.2.1.3.4 Terminal protein (TP) ................................................................................13 1.2.1.3.5 IVa2 ............................................................................................................13 1.2.1.3.6 Protease (adenain) ......................................................................................14 1.2.5 Life cycle of human adenoviruses........................................................................14 1.2.5.1 Cell entry ..........................................................................................................14 1.2.5.2 Transit of adenovirus to the nucleus ................................................................18 1.2.5.3 Import of species C adenoviruses into the nucleus ..........................................19 1.2.6 The genome of species C adenoviruses ...............................................................19 1.2.6.1 Early gene expression in species C adenoviruses ............................................21 iv 1.2.6.1.1 E1A ............................................................................................................21 1.2.6.1.2 E1B ............................................................................................................23
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