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Viewed in Pettersson and Robert, 1986) MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Sumithra Jayaram Candidate for the Degree: Doctor of Philosophy ______________________________________________________________ Dr. Eileen Bridge, Director ______________________________________________________________ Dr. Gary Janssen, Reader ______________________________________________________________ Dr. Anne Morris Hooke _______________________________________________________________ Dr. Mary E. Woodworth _______________________________________________________________ Dr. Qingshun Quinn Li, Graduate School Representative ABSTRACT INVESTIGATING ADENOVIRUS INTERACTIONS WITH HOST DOUBLE- STRAND BREAK REPAIR DEFENSES By Sumithra Jayaram The goal of this study was to investigate the role of host double-strand break repair (DSBR) on the life cycle of Adenovirus (Ad). Ad mutants that lack the entire E4 region activate a cellular DNA damage response accompanied by phosphorylation of several host DSBR and DNA damage response proteins. We find that aspects of the E4 mutant induced DNA damage response occurs at the onset of viral DNA replication and may be activated by physical replication of viral genomes. Genetic analysis of the E4 mutants revealed that the E4-34kDa protein was required to prevent the activation of DNA damage response. Redistribution of MRN complex proteins away from viral replication centers by the E4-11kDa protein was not sufficient to prevent the DNA damage response. Activation of the DNA damage response does not interfere with viral DNA replication in the presence of E4-11kDa protein. E4 mutants are severely defective for late gene expression following concatenation of their genomes by host DSBR proteins. We find that E4 mutant late gene expression improves in MO59J cells that fail to form genome concatemers. DSBR kinase inhibitors interfere with genome concatenation and also stimulate late gene expression. Concatenation of E4 mutant genomes interferes with cytoplasmic accumulation of viral late messages and leads to reduced late protein levels and poor viral yields following high multiplicity infection. However, failure to concatenate viral genomes did not rescue either the DNA replication defect or virus yield following low multiplicity E4 mutant infection. Our results indicate that if the E4 mutant DNA replication defect is overcome by high multiplicity infection, concatenation of the replicated genomes by host DSBR interferes with viral late gene expression. These studies provide insight into the role of host DSBR as an obstacle to a productive Ad infection, and how the virus dismantles this barrier. INVESTIGATING ADENOVIRUS INTERACTIONS WITH HOST DOUBLE-STRAND BREAK REPAIR DEFENSES A DISSERTATION Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Sumithra Jayaram Miami University Oxford, Ohio 2005 Dissertation Director: Dr. Eileen Bridge Table of Contents Page INTRODUCTION…………………………………………………………….. 1 Adenovirus life cycle…………………………………………………………… 1 Role of E4 region in Ad life cycle……………………………………………… 5 Role of host DSBR proteins in the Ad life cycle……………………………….. 7 E4 proteins interfere with cellular DNA damage response and prevent genome concatenation…………………………………………………………………… 16 CHAPTER 1: Investigating the induction of the cellular DNA damage response by an Adenovirus E4 mutant……………………………………………………… 18 Abstract………………………………………………………………………….. 19 Introduction……………………………………………………………………… 20 Materials and Methods…………………………………………………………… 23 Results…………………………………………………………………………… 25 Discussion……………………………………………………………………….. 45 CHAPTER 2: Genome concatenation contributes to the late gene expression defect of an Adenovirus E4 mutant……………………………………………….. 49 Abstract………………………………………………………………………. 50 Introduction………………………………………………………………….. 51 Materials and Methods………………………………………………………. 54 Results……………………………………………………………………….. 57 Discussion…………………………………………………………………… 77 CONCLUDING REMARK……………………………………………….. 82 Activation of the DNA damage response affects viral DNA replication and gene expression………………………………………………………………….. 86 REFERENCES………………………………………………………………. 95 ii List of Figures Page Figure 1 Adenovirus genome organization and detailed map of the E4 2 transcription unit. Figure 2 Schematic representation of the cellular response to DNA DSBs. 9 Figure 3 Schematic representation of the pathway of DNA NHEJ in 14 eukaryotes. Figure 4 E4 mutant does not activate a DNA damage response at low 26 multiplicities. Figure 5 E1a- Ad β-gal does not induce a DNA damage response. 29 Figure 6 E4 mutant-induced DNA damage response correlates with the 32 onset of viral DNA replication. Figure 7 Redistribution of the MRN complex members by E4 11kDa 37 protein does not prevent the activation of a DNA damage response. Figure 8 The activation of the DNA damage response does not affect 40 H5dl1010 DNA replication. Figure 9 DNA-PKcs is not required for the activation of DNA damage 43 signaling. Figure 10 E4 mutant late protein synthesis improves in MO59J cells that 58 lack DNA-PKcs. iii Figure 11 Inhibition of DSBR kinases interferes with E4 mutant genome 61 concatenation. Figure 12 Inhibition of DSBR kinase activity partially rescues the late 64 protein defect of an E4 mutant virus. Figure 13 The effect of DSBR kinase inhibitors on the DNA damage 67 response induced by E4 mutant infections. Figure 14 E4 mutant genome concatenation interferes with viral late 70 mRNA accumulation. Figure 15 The DNA replication defect of E4 mutants at low MOI is 74 not rescued by the failure to concatenate viral genomes. Figure 16 A model for the role of host DSBR proteins on the life 93 cycle of an Adenovirus E4 mutant. iv List of Tables Page Table 1 Summary of the genotype of E4 mutants used in this study 36 Table 2 Virus yield following high and low multiplicity infection 76 of MO59J and MO59K cells. v Acknowledgements I would like to express my deepest gratitude to my advisor, Dr. Eileen Bridge, for her excellent guidance, and support over the past years. Your are an amazing role model for women in science. Throughout my doctoral work you have encouraged me to develop independent thinking and research skills. Thank you for teaching me to be good scientist and greatly assisting me with scientific writing. I am also very grateful for having an exceptional doctoral committee and wish to thank Dr. Gary Janssen, Dr. Anne Morris Hooke, Dr. Mary E. Woodworth and Dr. Qinshun Li for their continual support and encouragement. I would specially like to thank Dr. Janssen for his words of wisdom during bleaker times when I needed to hear them. Thank you for always believing in me and encouraging me to do well in my scientific career. Thank you to my current lab mate Shomita Mathew. I have enjoyed working with you and wish you all the very best in your scientific pursuits. I also wish to thank my past lab mates Arunima and Kara Corbin-Lickfette, for their support during my initial phases of doctoral studies. I would like to thank the people of the department of microbiology for all your help in the past years. I will always be eternally be grateful for the opportunity given to me to come all the way from India to pursue my doctoral degree. Finally, I would like to thank my mom and dad, to whom I dedicate this dissertation. It was their dream and I am very happy to be able to fulfill this for them. Last but definitely not the least, my heartfelt thanks to my loving husband, Pradeep Dinakar, without whose support I would have not be able to complete my doctoral studies successfully. I hope I can always be equally supportive of your goals in life. vi INTRODUCTION Adenovirus (Ad) is used extensively as a model system for the study of DNA replication, gene expression and cell transformation. Ad is a double-stranded DNA virus whose replication cycle is largely dependent on the activity of cellular factors. The virus life cycle is divided into early and late phases separated by the onset of viral DNA replication. A set of early proteins initiates DNA replication, which is required for viral late gene expression. During the late phase, host cell machinery is used for the production of viral late messages and the structural proteins that associate with the viral DNA to form mature viral particles in the nucleus of the infected cells (reviewed in Pettersson and Robert, 1986). A. Adenovirus life cycle: Early phase. Adenovirus attaches with high efficiency to cellular receptors via the fiber protein (Chroboczek et al., 1995). The primary receptor for the human Ad is identical to that for coxsackie B virus and has therefore been termed the coxsackie-adenovirus receptor (CAR) (Tomko et al., 2000). The early phase of infection is characterized by the production of more than 20 regulatory proteins encoded by five early transcription units: early region 1a (E1a), E1b, E2, E3 and E4 (Fig. 1). Each unit produces multiple differentially spliced mRNAs encoding a variety of distinct polypeptides that are required to establish an optimal environment for efficient viral DNA replication and subsequent expression of viral late genes. After the virus uncoats and its genome enters the nucleus, the E1a promoter is immediately recognized by the cellular transcription machinery. The E1a gene products are required for efficient transactivation from the other early promoters (E1b, E2, E3 and E4). The E1a products also deregulate normal cell cycle control allowing quiescent cells to
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