A functional analysis of the non-coding RNAs of murine gammaherpesvirus 68 Nila Roy Choudhury Submitted for the degree of Doctor of Philosophy The University of Edinburgh 2010 Declaration I declare that this thesis has been composed by me and that all work included in this thesis is my own except where otherwise stated. No part of this work has been submitted, or will be submitted for any other degree of professional qualification. Nila Roy Choudhury 2010 Centre for Infectious Diseases The Roslin Institute and Royal (Dick) School of Veterinary Studies University of Edinburgh Summerhall Edinburgh EH9 1QH ii Acknowledgments I would like to thank my supervisors Dr Bernadette Dutia, Dr Simon Talbot and Professor Alastair Aitken for their help and advice during my PhD. As my main advisor Dr Bernadette Dutia provided guidance and advice at every stage of my work and I am very grateful to have been given the opportunity to do my PhD in her research group. A big thank you also to Dr Andrew Cronshaw for all his help with the cICAT technique and Amy Buck for her help with the qRT-PCR for miRNAs and general advice. I would also like to thank the rest of the people in the group and all the people in the tower block for creating a fantastic working environment. Thank you to my family, especially my parents who always encouraged me to work for what I wanted and not give up. Finally, Ewan, thank you for being there supporting me throughout my PhD, encouraging me and believing in me. iii Abstract Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in iv reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data. v Contents Title i Declaration ii Acknowledgments iii Abstract iv Contents vi List of Figures xi List of Tables xiv Abbreviations xv 1. Chapter One: Introduction 1 1.1. Herpesviruses 2 1.1.1. Classification 2 1.1.1.1. Alphaherpesvirinae 4 1.1.1.2. Betaherpesvirinae 4 1.1.1.3. Gammaherpesvirinae 4 1.1.2. Herpesvirus structure 5 1.1.3. Herpesvirus genome 7 1.1.4. Herpesvirus life cycle 9 1.1.4.1. Entry 9 1.1.4.2. Gene expression 12 1.1.4.3. Replication 12 1.1.4.4. Assembly and egress 13 1.1.4.5. Latency 13 1.2. Gammaherpesviruses 14 1.2.1. Epstein-Barr virus 14 1.2.1.1. EBV latency 15 1.2.1.1.1. Latency transcripts 17 1.2.1.2. EBV infection and associated diseases 19 1.2.2. Kaposi’s sarcoma-associated herpesvirus 22 1.2.2.1. KSHV genome 23 1.2.2.2. KSHV epidemiology 23 1.2.2.3. KSHV latency 24 1.2.2.3.1. Latency transcripts 25 1.2.2.4. KSHV infection and associated diseases 27 1.2.2.4.1. Kaposi’s sarcoma 27 1.2.2.4.2. Primary effusion lymphoma 28 1.2.2.4.3. Multicentric Castleman’s disease 28 1.3. Murine gammaherpesvirus-68 29 1.3.1. Discovery and classification 29 1.3.2. MHV-68 virion 30 vi 1.3.3. MHV-68 genome 30 1.3.4. MHV-68 life cycle 31 1.3.4.1. Entry 31 1.3.4.2. Gene expression 31 1.3.4.3. Viral DNA replication 33 1.3.4.4. Assembly and egress 34 1.3.5. MHV-68 primary infection 34 1.3.6. MHV-68 latency 35 1.3.6.1. Latency in vitro 36 1.3.6.2. Latency transcripts 37 1.3.7. MHV-68 evasion of the host’s immune system 38 1.3.8. MHV-68 pathogenesis 38 1.3.9. MHV-76 and other related viruses 39 1.3.10. Left-hand end of MHV-68 40 1.3.10.1. M1 40 1.3.10.2. M2 42 1.3.10.3. M3 43 1.3.10.4. M4 43 1.3.10.5. vtRNAs and miRNAs 44 1.4. Non-coding RNAs 45 1.4.1. Viral non-coding RNA molecules 45 1.4.1.1. Alphaherpesvirus non-coding RNA molecules 45 1.4.1.1.1. HSV LATs 45 1.4.1.2. Gammaherpesvirus non-coding RNA molecules 47 1.4.1.2.1. EBV EBERs 47 1.4.1.2.2. HVS U RNAs 50 1.4.1.2.3. KSHV PAN RNAs 51 1.4.1.3. Adenovirus non-coding RNAs 52 1.4.1.3.1. VAI and VAII 52 1.4.2. tRNAs 53 1.4.2.1. tRNA structure 53 1.4.2.2. tRNA expression 55 1.4.2.3. tRNA functions 56 1.4.2.3.1. Translation 56 1.4.2.3.2. Amino acid starvation 56 1.4.2.4. tRNA genes and chromatin 57 1.4.2.5. tRNA functions during viral infections 58 1.4.2.6. tRNA-like molecules 58 1.4.2.6.1. Plant virus tRNA-like molecules 59 1.4.2.6.2. Transfer messenger RNA 59 1.4.2.6.3. The threonyl-tRNA synthetase gene of E.coli 60 1.4.3. miRNAs 60 1.4.3.1. miRNA biogenesis 60 1.4.3.2. miRNA functions 63 1.4.3.3. Viruses and miRNAs 64 1.5. Project outline 69 vii 2. Chapter Two: Materials and methods 70 2.1. Cell Culture techniques 71 2.1.1. Maintenance of cell lines 71 2.1.2. Harvesting and counting cells 71 2.1.3. Cytospins 71 2.1.4. Transfection 72 2.2. Virological methods 72 2.2.1. Preparation of virus stocks 72 2.2.2. Virus titration 73 2.2.3. Infection of NS0 cells for virus characterisation studies 73 2.2.4. Infective centre assay 74 2.2.5. GFP labelled viruses 74 2.2.6. Cloning of infected cells 74 2.2.7. Staining for lytic proteins 75 2.3. In situ hybridisation 75 2.3.1. Generation of labelled RNA probe 75 2.3.2. In situ hybridisation of cytospins 77 2.4. DNA isolation and manipulation 78 2.4.1. Plasmid preps 78 2.5. RNA isolation and manipulation 79 2.5.1. microRNA isolation 79 2.5.2. DNase treatment of RNA 79 2.5.3. Reverse transcription of RNA 79 2.5.4. In vitro transcription of vtRNAs 80 2.6. Polymerase chain reaction (PCR) 80 2.6.1. Standard PCR 80 2.6.2.