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Lora Grainger Dissertation 6 23 10 Characterization of the Transcripts that Encode pUL138, a Latency Determinant, During Human Cytomegalovirus Infection Item Type text; Electronic Dissertation Authors Grainger, Lora Ann Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 24/09/2021 15:04:11 Link to Item http://hdl.handle.net/10150/195915 1 CHARACTERIZATION OF THE TRANSCRIPTS THAT ENCODE PUL138, A LATENCY DETERMINANT, DURING HUMAN CYTOMEGALOVIRUS INFECTION by Lora A. Grainger Copyright © Lora A. Grainger 2010 A Dissertation Submitted to the Faculty of the DEPARTMENT OF IMMUNOBIOLOGY In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2010 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Lora A. Grainger entitled: Characterization of the Transcripts that Encode pUL138, a Latency Determinant, During Human Cytomegalovirus Infection. We recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy. _____________________________________________________Date: 6/15/10 Dr. Nafees Ahmad _____________________________________________________ Date: 6/15/10 Dr. Lonnie Lybarger _____________________________________________________ Date: 6/15/10 Dr. Carol Dieckmann Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement _____________________________________________________Date: 6/15/10 Dissertation Director: Dr. Felicia Goodrum 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Lora A. Grainger 4 ACKNOWLEDGEMENTS There are many people I would like to thank that have contributed to my completion of the Ph.D. but I owe Dr. Felicia Goodrum special thanks as she ‘rescued’ me from a string of bad luck that would have left me wavering if she had not invited me to join her laboratory. With that, I owe a tremendous amount of gratitude to her as well as Alex Petrucelli and Mike Rak who have been instrumental as team players in the development of the internationally recognized Goodrum laboratory. The four of us truly became a family while the lab was being established. I’d also like to thank the current and past members of the Goodrum laboratory. Thank you for all of your support! Much of this work was done in collaboration with Louis Cicchini who has contributed greatly to the identification of the HCMV IRES. He has become an extraordinary scientist and I am eternally grateful for his insight, enthusiasm and organizational skills through the duration of our time together in the laboratory. I would also like to thank the faculty and graduate students of the Department of Immunobiology for all of their support through this process, in particular, the late Dr. Deluca and Dr. Friedman who allowed me to work in their laboratories. Further, I am sincerely grateful for the friendship, support and knowledge extended by the Immunobiology graduate students during times of extreme happiness as well as when I needed them the most. Thank you! Throughout my time as a graduate student I have enjoyed sharing my love of science with others. Therefore, I would like to extend thanks to Nadja Anderson and Stacey Forsyth who have been instrumental in allowing me to extend my research to others through various biotechnology outreach initiatives. Thanks! Last but definitely not least, I would like to thank the members of my family and close friends for their support through the pursuit of my Ph.D. I’d especially like to thank my mom and dad who have provided unconditional love, support and independence that has made me who I am today! Thank you! 5 DEDICATION To my loving parents, John and Marylin Grainger. 6 TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………...…10 LIST OF FIGURES…………………………………………………………………….11 ABSTRACT…………………………………………………………………………….14 CHAPTER 1 Literature Review………………………………………………………15 1.1 Human Cytomegalovirus ………………………………………………...15 1.2 HCMV Latency…………………………………………………………….21 1.3 Mechanisms of Translation Initiation……………………………………31 1.4 IRESs……………………………………………………………………….36 1.5 The Type I Interferon Response…………………………………………40 1.6 Type I IFN Response and HCMV Latency…………………………......44 CHAPTER 2 Materials and Methods………………………………………………...46 2.1 Cells………………………………………………………………………...46 2.2 Viruses……………………………………………………………………...47 2.3 Plasmids……………………………………………………………………48 2.4 Transient Transfection and Luciferase Reporter Assays………….….50 2.5 Immunoblotting………………………………………………………….…51 2.6 Northern Blotting…………………………………………………………..52 2.7 RACE……………………………………………………………………….53 2.8 In Vitro Transcription and Translation…………………………………..55 2.9 RNA Structure Prediction………………………………………………...55 7 TABLE OF CONTENTS-Continued 2.10 qRT-PCR Transcript Length Analysis…………………………………56 2.11 qRT-PCR IE, UL138 Expression and IFN Analysis………………….58 2.12 Interferon Manipulation………………………………………………….60 CHAPTER 3 UL138 transcript expression during productive and latent Human cytomegalovirus infection……………………………………………..63 3.1 Introduction………………………………………………………………...63 3.2 UL138 is encoded on multiple transcripts in fibroblasts and hematopoietic progenitor cells……………………………………….65 3.3 UL138 transcripts are specific and are present in FIX∆UL138STOP infected fibroblasts…………………………………68 3.4 The UL138 transcripts in FIX∆UL138STOP infected cells are stable…………………………………………………………70 3.5 Kinetics of UL138 expression in fibroblasts and CD34+ HPCs……………………………………………………………………71 3.6 Discussion and Future Directions……………………………………….74 CHAPTER 4 Protein coding capacity of UL138 transcripts……………………….76 4.1 Introduction………………………………………………………………...76 4.2 Putative ORFs upstream of UL138 encode proteins during HCMV infection……………………………………………...78 4.3. pUL138 is expressed from polycistronic mRNAs……………...……...81 4.4 Model of the coding potential of the polycistronic UL138 transcripts..84 8 TABLE OF CONTENTS-Continued 4.5 Discussion and Future Directions……………………………………….85 CHAPTER 5 pUL138 is expressed by alternative mechanisms of translation initiation………………………………………………….…88 5.1 Introduction…………………………………………………………….…..88 5.2 A sequence overlapping the 5′ end of the UL136 ORF promotes translation of a downstream cistron………………….…...91 5.3 Transcripts originating from the dual luciferase vector are full length and ICS-7 do not have inherent promoter activity………..…94 5.4 ICS-7 Mfold prediction suggests extensive RNA secondary structure…………………………………………………….98 5.5 ICS-7 IRES activity is dependent on eIF4G…………………………..100 5.6 Discussion and Future Directions……………………………………...101 CHAPTER 6 pUL138 expression from the 3.6- and 2.7-kb transcripts is stress inducible……………………………………….104 6.1 Introduction……………………………………………………………….104 6.2 ICS-7 is a stress-inducible IRES……………………………………….106 6.3 UL138 cistron expression from the 3.6- and 2.7-kb transcripts, but not the 1.4-kb transcript, is stress-inducible…………………..107 6.4 IRES mediated UL138 cistron expression from the 2.7-kb transcript is dependent on eIF4G………………….…..111 9 TABLE OF CONTENTS-Continued 6.5 UL138 protein expression is stress-inducible from the 3.6- and 2.7-kb UL138 transcripts, but not the 1.4-kb transcript…………..113 6.6 Discussion and Future Directions……………………………………...116 CHAPTER 7 The role of type I IFN in human cytomegalovirus latency………..119 7.1 Introduction……………………………………………………………….119 7.2 The ULb′ ORFs UL136-UL142 are associated with decreased resistance to the type I IFN response in MRC5 fibroblasts……...121 7.3 The ULb′ ORFs UL136-UL142 are associated with decreased resistance to IFNα challenge…………………………..124 7.4 The ULb′ ORFs UL136-UL142 are associated with increased resistance to the type I IFN response in CD34+ HPCs………….126 7.5 ORFs UL136-UL142 are necessary for FIX to inhibit ISG expression in CD34+ HPCs…………………………………………129 7.6 Discussion and Future Directions……………………………………...132 CHAPTER 8 Discussion……………………………………………………………..136 REFERENCES……………………………………………………………………….154 10 LIST OF TABLES Table 1 Primers used for cloning expression vectors……………………………...60 Table 2 Primers used for recombinant virus generation…………………………..62 11 LIST OF FIGURES Figure 1. Human cytomegalovirus virion organization…………………………….17 Figure 2. Genome organization of laboratory adapted and clinical strains of HCMV……………………………………………………….20 Figure 3. Classification of human herpesviruses…………………………………..22 Figure 4. in vitro HCMV latency model system………………………..…………..24 Figure 5. pUL138 promotes a latent infection in CD34+ hematopoietic progenitor cells……………………………………………….26 Figure
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