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Sharon Carr Mphil Thesis ADENOVIRUS AND ITS INTERACTION WITH HOST CELL PROTEINS Sharon Carr A Thesis Submitted for the Degree of MPhil at the University of St. Andrews 2007 Full metadata for this item is available in Research@StAndrews:FullText at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/219 This item is protected by original copyright Adenovirus and its interaction with host cell proteins Sharon Carr School of Biomedical Sciences University of St Andrews A thesis submitted for the degree of Master of Philosophy September 2006 1 I, …………………., hereby certify that this thesis, which is approximately 75,000 words in length, has been written by me, that it is the record of work carried out by me and that it has not been submitted in any previous application for a higher degree. date…………… signature of candidate………………………… I was admitted as a research student in September 2002 and as a candidate for the degree of Doctor of Philosophy in September 2002; the higher study for which this is a record was carried out in the University of St Andrews between 2002 and 2005, and at the University of Dundee between 2005 and 2006. date…………… signature of candidate………………………… I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriated for the degree of Master of Philosophy in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. date…………… signature of supervisors………………………… ………………………… 2 In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and abstract will be published, and that a copy of the work may be made and supplied to any bona fida research worker. date…………… signature of candidate………………………… 3 CONTENTS Declaration……………………………………………………………………2 List of figures…………………………………………………………………16 List of tables…………………………………………………………………..18 List of abbreviations…………………………………………………………..19 Acknowledgments…………………………………………………………….27 ABSTRACT………………………………………… ……………………..28 1. INTRODUCTION…………………………………………………………29 1.1 Adenoviruses……………………………………………………………29 1.1.1 Adenoviruses…………………………………………………………..29 1.1.2 The family adenoviridae……………………………………………….29 1.1.3 Human Adenoviruses…………………………………………………..30 1.1.4 Virion structure………………………………………………………...31 1.1.5 Genome organisation…………………………………………………..34 1.2 E1A region………………………………………………………………35 1.2.1 The E1A region………………………………………………………...35 1.2.2 E1A binds p21 and related CDK inhibitors thereby stimulating cell division and growth…………………………………………………..36 1.2.3 E1A binds to cyclins A and E-CDK complexes, which regulate passage into cell DNA synthesis…………………………………………..…37 4 1.2.4 E1A binds to the p300 / CBP family of transactivators and has numerous effects…………………………………………………………………39 1.2.5 E1A binds to Rb / p130 family of proteins and in doing so thus releases E2F……………………………………………………………….…..42 1.2.6 E1A binds to TATA-box-binding protein (TBP) and TBP associated proteins……………………………………………………..………..43 1.2.7 E1A is required for transformation……………………………………..44 1.2.8 The E1A proteins induce apoptosis through a p53-dependent pathway.45 1.2.9 The E1A region also induces apoptosis via a p53-independent pathway.46 1.3 The E1B region 1.3.1 Proteins produced by the E1B region and their localisation patterns..47 1.3.2 E1B-19kDa protein…………………………………………………..47 1.3.3 The E1B55kDa protein is required for efficient shutoff of host cell replication…………………………………………………………….48 1.3.4 E1B-55kDa and E4orf6 proteins interact……………………………..49 1.3.5 The E1B-55kDa protein facilitates the cytoplasmic accumulation of late region viral mRNAs during infection…………….………………...49 1.3.6 E4 Orf6 causes relocalisation of E1B-55kDa protein from the cytoplasm to the nucleus………………………………………………………....51 1.3.7 E4 Orf6 and E1B-55kDa proteins form a complex and shuttle between the nucleus and the cytoplasm…………………………………………..….52 1.3.8 E1B-55kDa protein can shuttle between the nucleus and the cytoplasm independently of E4 Orf6……………….……………………………….52 5 1.3.9 E4 Orf3 also relocalises E1B-55kDa protein from the cytoplasm to the nucleus………………………………………………………………….53 1.3.10 Identification of cellular proteins that interact with E1B-55kDa protein.55 1.3.11 The E1B55-kDa protein is phosphorylated……………………………55 1.3.12 The E1B55kDa protein interacts with p53………………………….....56 1.3.13 E1B-55kDa protein regulates the levels of p53 protein……………..….58 1.3.14 E1B-55kDa protein acts as a transcriptional repressor of p53……….....58 1.3.15 E1B-55kDa protein has no effect on the p53 related protein p73……...59 1.3.16 The E1B55-kDa protein inhibits p53 acetylation by PCAF………..…...60 1.3.17 The E1B-55kDa protein interacts with a mSin3A/Histone deacetylase complex………………………………………………….…60 1.3.18 Degradation of p53 by E1B-55kDA and E4 Orf6 proteins occurs via a novel mechanism involving a Cullin-containing complex……………...61 1.3.19 E1B-55kDa protein interacts with Daxx………………..………….…….63 1.4 The E2 region…………………………………………………………..…63 1.5 The E3 region………………………………….…………………………..64 1.6 The E4 region……………………………………………………………..66 1.6.1 The E4 region encodes 7 different proteins…………………..………66 1.6.2 The E4 region is required for efficient DNA replication, late gene expression and shutoff of synthesis of host cell proteins…………….66 1.6.3 E4 Orf1………………………………………………………………67 6 1.6.4 E4 Orf2……………………………………….………………………68 1.6.5 The E4 Orf3 and E4 Orf6 proteins can compensate for each other..…68 1.6.6 E4 Orf4…………………………………………………………………70 1.6.7 E4 Orf3/4……………………………………………………………….70 1.6.8 E4 Orf6/7……………………………………………………………….71 1.7 Intermediate and late genes………………………………………………71 1.8 Viral replication……………………………………………………………72 1.9 Adenovirus as a viral vector……………………………………………….73 1.9.1 Why are people interested in studying adenoviruses?..............................73 1.9.2 Adenovirus as a gene therapy vector……………………………….…..74 1.9.3 Adenovirus in cancer therapy……………………………………….….74 1.10 Small Ubiquitin Modifying protein (SUMO)………………………….75 1.10.1 Introduction to SUMO……………………….………………………....75 1.10.2 Viral proteins that are modified by SUMO…………………….………80 1.10.3 The Cytomegalovirus immediate-early 1 (CMV-IE1) and immediate early 2 proteins (CMV-IE2) are modified by SUMO…………………………81 1.10.4 The papillomavirus E1 (PV-E1) protein is modified by SUMO………..82 1.10.5 The Epstein-Barr virus immediate-early protein BZLF1 (EBV-Z) is modified by SUMO………………………………………………….......83 7 1.10.6 The human herpes virus 6 immediate-early 1 (HHV6-IE1) protein is modified by SUMO………………………………………………….......83 1.10.7 The adenovirus E1B-55kDa protein is modified by SUMO……..….…..84 1.11 Proteomics methods for studying protein interactions……………..86 1.11.1 Proteomics methods for studying adenoviruses……………………..86 1.11.2 Immunoprecipitation………………………………………………….88 1.11.3 Yeast II Hybrid………….…………………………………………….89 1.11.4 Tandem Affinity Purification…………………………………………..89 1.11.5 Mass Spectrometry……………………………………………………..90 1.11.6 Aim of this work………………………………………………………97 2. MATERIALS AND METHODS………………………………………….99 2.1 DNA Cloning and analysis…………………………….………………….99 2.1.1 Polymerase chain reaction………………………….…………………..99 2.1.2 Clean-up of DNA after PCR…………………………….…………...…99 2.1.3 Agarose gel electrophoresis…………………….………………………99 2.1.4 Purification of DNA from agarose gels………………………………...99 2.1.5 Restriction digest of DNA for cloning……………………….…………100 2.1.6 Dephosphorylation of linearised vector DNA………………………….100 2.1.7 Ligation of DNA………………………………………………………..100 2.1.8 Bacterial strains……………………………………………………….100 2.1.9 Preparation of transformation competent E. coli………………………101 2.1.10 Preparation of electrocompetent E. coli………………………………101 8 2.1.11 Transformation of transformation competent E. coli…………………102 2.1.12 Electroporation of electrocompetent E. coli…………………………102 2.1.13 Media used for E. coli…………………………………………………103 2.1.14 Purification of plasmid DNA………………………………………….103 2.1.15 Purification of plasmid DNA using alkali lysis miniprep method……104 2.1.16 Purification of plasmid DNA using alkali lysis maxiprep method…..105 2.1.17 Quantification of DNA………………………………………….……105 2.1.18 Phenol chloroform purification and ethanol precipitation of DNA….105 2.1.19 Use of radiolabelled probes for the detection of positive colonies…….106 2.1.20 DNA sequence analysis………………………………………………108 2.2 Cell culture…………………………………………………………….108 2.2.1 Cell culture……………………………………..……………………108 2.2.2 Freezing down cells………………………………………………….108 2.2.3 Resuscitating cells from liquid nitrogen……………………………...108 2.2.4 Transient transfection of cells……………………………….………109 2.2.5 Immunofluorescence……………………………………………….….109 2.2.6 Making recombinant adenoviruses……………………………………110 2.2.7 Isolation of recombinant viruses by end point dilution………………111 2.2.8 Screening of recombinant adenoviruses………………………………111 2.2.9 Extraction of viral DNA using the Qiagen QIAmp DNA Blood minikit …………………………………………………………………………112 2.2.10 Amplification of recombinant viruses…………………………..……113 2.2.11 Infecting HeLa spinners with adenovirus…………………………113 9 2.2.12 Arcton extraction of adenovirus…………………………………..113 2.2.13 Virus purification by CsCl gradient……………………………….114 2.2.14 Titration of virus stocks……………………………………………115 2.2.15 Experiment to determine the optimum concentration of puromycin required to kill HeLa cells……………….…………………………115
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