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Adenovirus Type 40 Host Range in Tissue Culture: Replication and Gene Expression in INT407 Cells

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Adenovirus Type 40 Host Range in Tissue Culture: Replication and Gene Expression in INT407 Cells Adenovirus Type 40 Host Range in Tissue Culture: Replication and Gene Expression in INT407 Cells by Robina R. Ullah A Thesis Presented for the Degree of Doctor of Philosophy in The Faculty of Science at the University of Glasgow. Institute of Virology, Church Street, Glasgow, Gil 5JR. January, 1997. ProQuest Number: 13815377 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13815377 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 /0 < M TABLE OF CONTENTS. Summary. Acknowledgements. Abbreviations. 1. INTRODUCTION. 1 1.1. The Adenoviridae. 1 1.1.1. Classification. 1 1.1.2. Morphology. 1 1.1.3. Genome Structure. 3 1.1.4. Lytic Cycle. 4 1.1.4.1. Viral Entry into Cells. 4 1.1.4.2. Transcription. 5 1.1.4.3. DNA Replication. 13 1.1.4.4. Virion Assembly. 15 1.2. Attachment and Internalisation. 16 1.2.1. Attachment. 16 1.2.2. Internalisation. 18 1.2.2.1. Penton Base Protein. 18 1.2.2.2. Integrins have a Role in Adenovirus Entry. 19 1.2.2.3. Role of av Integrins in Other Adenovirus Serotypes. 20 1.2.2.4. av Integrins Enhance Adenovirus Mediated Gene-Delivery. 21 1.2.2.5. Post-Internalisation Events. 21 1.3. Early Region 1A. 23 1.3.1. El A Transcription. 23 1.3.2. El A Proteins. 24 1.3.3. Transactivation. 25 1.3.4. Transcription Repression. 29 1.3.5. Transformation. 29 1.4. Early Region IB. 32 1.4.1. Genomic Organisation of the E1B Region. 32 1.4.2. E1B Transcription. 33 1.4.3. E1B Proteins. 34 1.4.4. E1B Promoter. 34 1.4.5. The 19K Protein. 36 1.4.6. The 55K Protein and Related Polypeptides. 40 1.5. Enteric Adenoviruses. 45 1.5.1 Identification and Classification. 45 1.5.2 Epidemiology. 46 1.5.3. Pathogenesis. 46 1.5.4. The Molecular Biology of the Enteric Adenoviruses. 47 1.5.4.1. Early Regions. 48 1.5.4.2. Late Regions. 50 1.5.5 Growth Properties of Enteric Adenoviruses in Culture. 51 1.5.6. Aims of Project. 55 2. MATERIALS. 56 2.1. Tissue Culture. 56 2.1.1. Tissue Cells. 56 2.1.2. Tissue Culture Media 56 2.2. Viruses. 57 2.3. Bacteria 58 2.3.1. Bacterial Strain. 58 2.3.2. Bacterial Culture Media. 58 2.4. Plasmids. 58 2.5. Oligonucleotides. 59 2.6. Enzymes. 60 2.7. Antibodies. 60 2.8. Chemical Reagents. 61 2.9. Radiochemicals. 61 2.10. Solutions. 61 3. METHODS. 64 3.1. Tissue Culture. 64 3.2. Virus Preparation. 64 3.2.1. Virus Stocks. 64 3.2.2. Density-Gradient Purified Virus. 65 3.2.3. Labelled Virus. 66 3.3. Extraction of Nucleic Acids. 66 3.3.1. Extraction of Viral DNA. 66 3.3.2. Extraction of Intranuclear Viral DNA. 67 3.3.3. Preparation of Cytoplasmic RNA. 67 3.4. DNase I Treatment of RNA. 68 3.5. Quantitation of Nucleic Acid. 68 3.6. Electrophoretic Separation of Nucleic Acids. 69 3.6.1. Agarose 'Midi' Gel. 69 3.6.2. Formaldehyde Agarose Gels. 69 3.6.3. Non-denaturing Polyacrylamide Gels. 69 3.6.4. Denaturing Urea/Polyacrylamide Gels. 70 3.7. Electroelution of DNA from Polyacrylamide Gels. 70 3.8. Preparation of Plasmid DNA. 70 3.8.1. Small Scale Preparation of Plasmid DNA. 70 3.8.2. Large Scale Preparation of Plasmid DNA. 71 3.9. Radioactive Labelling of DNA. 72 3.9.1. Internal Labelling of DNA by Nick Translation. 72 3.9.2. 5' End Labelling of Oligonucleotides. 72 3.10. Manipulation of Oligonucleotides. 73 3.10.1. Purification of Oligonucleotides. 73 3.10.2. Annealing Complementary Oligonucleotides. 73 3.11. DNA Slot Blot Analysis. 74 3.12. Northern Blot Analysis. 75 3.13. SI Nuclease Digestion. 76 3.14. Indirect Immunofluorescence. 76 3.15. Preparation of Crude Ad2 Fibre. 77 3.16. Attachment and Internalisation Assay. 78 3.17. Preparation of Nuclear Extracts. 79 3.18. WGA Affinity Chromatography of Spl. 79 3.18.1. Preparation of Spl extracts. 79 3.18.2. WGA Affinity Chromatography. 80 3.19. Gel Retardation Assay. 80 4. RESULTS. 82 4.1. Timecourse of Viral DNA Replication in Ad40 Infected Permissive and Semi-Permissive Cells. 84 4.1.1. Summary. 85 4.2. Replication of Ad5 and Adenovirus Early Region El Mutants on Permissive and Semi-Permissive Cells. 86 4.3. Growth Kinetics of Ad40 on Permissive and Semi-Permissive Cells. 90 4.3.1. Investigation of the Growth Kinetics of Ad40 on Permissive and Semi- Permissive Cell Lines Using Indirect Immunofluorescence. 91 4.3.2. Particle to Infectivity Ratio. 94 4.3.3. Summary. 94 4.4. Attachment and Internalisation of Ad40 and dl309 on INT407 and HeLa Cells. 96 4.4.1. Attachment of Ad40 and dl309 to INT407 and HeLa cells. 97 4.4.1.1. Attachment of Ad40 to INT407 and HeLa Cells. 98 4.4.1.2. Attachment of dl309 to INT407 and HeLa Cells. 99 4.4.1.3. Competition of Attached Virus with Ad40 Virus or Ad2 Fibre. 100 4.4.1.4. Summary. 101 4.4.2. Analysis of Adenovirus Internalisation. 101 4.4.2.1. Internalisation of Ad40 in INT407 and HeLa Cells. 102 4.4.2.2. Internalisation of dl309 in INT407 and HeLa Cells. 103 4.4.2.3. Summary. 103 4.5. Analysis of El Transcription in Ad40 Infected Cells. 104 4.5.1. A Timecourse of Ad40 El Transcription and DNA Replication in Infected Cells. 105 4.5.1.1. Summary. 106 4.5.2. Analysis of E1B mRNA Produced in the Presence and Absence of DNA Replication. 107 4.5.2.1. Summary. 110 4.6. Analysis of the Ad40 E1B Promoter. 112 4.6.1. Analysis of the Ad40 and Ad2 GC Box Sequences by Gel Retardation Assay. 113 4.6.2. Gel Retardation Competition Assays of Ad40 and Ad2 GC Box Sequences. 115 4.6.3. Summary. 117 5. DISCUSSION. 119 5.1. INT407 Cells are Permissive for Ad40. 119 5.2. Ad40 Growth in Permissive Cells Shows One Hit Kinetics. 122 5.3. Ad40 Entry into Cells. 124 5.4. Ad40 El Gene Expression. 127 5.5. The Ad40 E1B Promoter. 130 5.6. Future Work. 133 REFERENCES List of Figures and Tables. Figure After page No. 1.1.1 a. A Schematic View of the Adenovirus Particle. 1 1.1.1 b. A Diagram of the Icosahedral Adenovirus Capsid. 1 1.1.2. Organisation of the Ad2 Genome. 4 1.1.3. Adenovirus Assembly Pathway. 15 1.3.1. Ad2 E1A mRNAs and Protein Products. 23 1.3.2. The Structural and Functional Domains of the El A Protein. 24 1.3.3. Diagram of Adenovirus Promoter Elements and Factor Binding Sites 25 1.4.1. Transcription Map for the Ad2 E1B Region. 32 1.5.1. Transcription Maps for Ad40 (Dugan) and Ad41 (Tak) E1B Regions. 49 4.1.1. Timecourse of Virion DNA Replication on Ad40 Infected KBa+b, ENT407 and HeLa Cells. 84 4.1.2. Quantitative Analysis of Ad40 DNA in Various Cell Lines. 84 4.2.1. Infection of Ad5/2 Mutants on KBa+b, HeLa, INT407 and A549 Cells. 87 4.2.2. Infection of Ad5/2 Mutants on KBa+b, HeLa, INT407 and A549 Cells at a Low moi. 88 4.2.3. Quantitation of Ad5/2 and Ad40 Virion DNA from Various Cell Lines. 88 4.3.1. Ad40 Fluorescent Cell Counts for Permissive and Semi-Permissive Cell Lines, at Different Input Concentrations. 92 4.3.2. Theoretical Lines for One and Two Hit Kinetics 93 4.4.1. Analysis of Ad40 Attachment to INT407 and HeLa Cells. 98 4.4.2. Analysis of dl309 Attachment to INT407 and HeLa Cells. 99 4.4.3. Comparison of Ad40 and dl309 Virus Attachment. 99 4.4.4. Attachment of Labelled Ad40 Virus is Inhibited by a Ten Fold Excess of Unlabelled Virus. 100 4.4.5. Competition of dl309 Virus with Ad2 Fibre. 100 4.4.6. Ad40 Internalisation in INT407 Cells. 102 4.4.7. Ad40 Internalisation in HeLa Cells. 102 4.4.8. dl309 Internalisation in INT407 Cells. 103 4.4.9. dl309 Internalisation in HeLa Cells. 103 4.5.1. A Timecourse of E1B Transcription and DNA Replication in KBa+b, HeLa and INT407 cells. 105 Figure After page No. 4.5.2. A Timecourse of El A Transcription and DNA Replication in KBa+b, HeLa and INT407 Cells. 106 4.5.3. Inhibition of DNA Replication by ara-C in Ad40 Infected Cells. 108 4.5.4. Analysis of Ad40 E1B Transcription in the Absence and Presence of ara-C in Permissive and Semi-Permissive Cells. 109 4.6.1. Alignment of Adenovirus E1B Promoter Sequences. 112 4.6.2. Gel Retardation Analysis of Ad2 and Ad40 GC Box Sequences with Different Extracts. 114 4.6.3. Competition of DNA-Protein Binding with the Ad2 Probe.
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