MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Anand Prakash Candidate for the Degree: Doctor of Philosophy Dr. Eileen Bridge, Mentor Dr. Gary R. Janssen, Reader Dr. Joseph M. Carlin, Reader Dr. Xiao-Wen Cheng Dr. David G. Pennock Graduate School Representative ABSTRACT INVESTIGATING THE TRIGGERS FOR ACTIVATING THE CELLULAR DNA DAMAGE RESPONSE DURING ADENOVIRUS INFECTION by Anand Prakash Cellular genomic integrity is constantly attacked by a variety of exogenous and endogenous agents. In response to damaged DNA, the cell activates a DNA damage response (DDR) pathway to maintain genomic integrity. Cells can also activate DDRs in response to infection with several types of viruses. The cellular DDR pathway involves sensing DNA damage by the Mre11, Rad50, Nbs1 (MRN) sensor complex, which activates downstream ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases. These kinases phosphorylate downstream effector proteins implicated in cell cycle arrest, DNA repair, and, if the damage is irreparable, apoptosis. The induction of DDRs includes focal accumulation and phosphorylation of several DDR proteins. Adenovirus (Ad) mutants that lack early region 4 (E4) activate a cellular DDR. E4 proteins normally inactivate the MRN sensor complex and prevent downstream DDR signaling involved in DNA repair and cell cycle checkpoint arrest in wild- type Ad5 infections. The characteristics of Ad infection that activate the cellular DDR are not well understood. We have investigated the ability of replication defective and replication competent Ad mutants to activate cellular DDRs and G2/M cell cycle arrest. Ad infection induced early focal accumulation of DDR proteins such as Mre11, Mdc1, phosphorylated ATM (pATM), phosphorylated Chk2 (pChk2), and 53BPI, independent of the replication status of the mutants studied. However, Mre11 and pATM foci were transient in replication defective infections and were only maintained in infections with replication competent mutants. Viral DNA replication was correlated with amplification of pATM levels as well as its substrates, pChk2 and pNbs1. Furthermore, we found that G2/M cell cycle arrest was not activated by a replication defective mutant or a mutant expressing the E4orf3 encoded 11kDa protein. Our results suggest that the initial induction of DDR foci does not require viral DNA replication. In contrast, viral DNA replication is important for maintenance of DDR proteins at viral replication centers, amplification of pATM, pChk2, and pNbs1, and G2/M cell cycle arrest. INVESTIGATING THE TRIGGERS FOR ACTIVATING THE CELLULAR DNA DAMAGE RESPONSE DURING ADENOVIRUS INFECTION 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 Anand Prakash Miami University Oxford, Ohio 2014 Dissertation Director: Eileen Bridge, Ph.D. Table of Contents List of Tables iii List of Figures iv Acknowledgements vi General Introduction 1 Chapter I. Differential activation of cellular DNA damage responses by 16 replication defective and replication competent adenovirus mutants Chapter II. Adenovirus DNA replication amplifies ATM dependent 56 signaling and contributes to the induction G2/M cell cycle arrest during activation of the DNA damage response Concluding Remarks 81 References 92 ii List of Tables Table 1. Status of E4-11kDa and E4-34kDa gene in the E4 22 mutants used in this study. iii List of Figures Figure 1. Diagrammatic representation of Adenovirus genome. 3 Figure 2. Model of Adenovirus DNA replication. 6 Figure 3. Cellular DNA Damage Response (DDR) activation. 10 Figure 4. Mdc1 focus formation in Ad-infected cells is correlated 27 with MOI. Figure 5. AdRSVβgal is defective for viral early gene expression 30 and viral DNA replication. Figure 6. Mdc1 focus formation depends on transcription from the 33 viral genome. Figure 7. Replication defective AdRSVβgal fails to activate phosphorylation 36 of Nbs1 and Chk1. Figure 8. Redistribution of Mre11 to nuclear tracks is not sufficient to prevent 39 Nbs1 phosphorylation. Figure 9. UV treatment of AdRSVβgal infected cells activates phosphorylation 42 of Nbs1. Figure 10. Viral DNA replication is important for activating Nbs1 phosphorylation. 45 Figure 11. E4 mutant-induced phosphorylation of Nbs1 and Chk1 correlates with the 48 onset of viral DNA replication. Figure 12. Focal accumulation of pATM is not maintained in the absence of DNA 66 replication or in the presence of E4-11kDa. Figure 13. ATM substrates are detected in foci in the absence of viral DNA 69 replication but an increase in phosphoprotein levels is not detected in western blot analysis. iv Figure 14. Viral DNA replication amplifies ATM dependent signaling. 72 Figure 15. G2/M cell cycle arrest occurs in 1007 but not in 1010 or E1- infections 75 Figure 16. Model for Ad induced cellular DDR. 89 v Acknowledgements I am grateful to my advisor Dr. Eileen Bridge for her guidance, support, and constant encouragement during my PhD carrier. I thank you for sincerely taking time out from your busy schedule to teach me to think in scientific manner. I deeply appreciated you correcting endless drafts of non-sequitur writing and guiding me to improve my scientific and thesis writing. You have been caring and supportive mentor in the matters non-scientific as well. I will miss talking to you about your animals. I thank my doctoral committee members who were instrument in my scientific growth. Dr Gary Janssen, thank you for your generous critiques on my manuscript and my dissertation. I appreciate you for your advice during the job negotiation, words of encouragement during my PhD, and philosophical conversations about the comic books. Dr. Joseph Carlin, thanks for reading and providing your valuable critiques on my dissertation. Also, your questions and comments during the various journal clubs, I have attended, enriched me scientifically. Dr. Xiao- Wen Cheng, thank you for providing guidance, suggestions, reagents, and access to your lab and office at any time. Dr. David Pennock, thank you for providing my valuable suggestions during my committee meetings. Thanks for being very accommodative. I thank people in the department of Microbiology who had helped me consciously and unconsciously over the years. Dipendra Gautam – I couldn’t ask for a better friend. Your memories have become inseparable from Oxford memories. You have taught me a lot and looking back, I see, I haven’t thanked you enough. Thank you! Jenna Dolhi –I don’t remember a single instance when I needed your help and you said no. Thanks for listening, helping, and being such a beautiful soul. Rachael Desmone – thoughtful, considerate, caring, and most down to earth I have met in USA. Thanks. Chris Sedlacek – an Indian friend outside of India. Thanks for all those conversations and dinners and making me feel home. Tyler Garretson and Nicole Marotta – you are rockstar TAs and wonderful human beings. Akhilesh Kumar, Ashwani Kumar, Subash Dhungana, and Amber Beckett – interacting with you people have always been blast. I wish you all the best in whatever journey you take. Barb and Darlene, thank you for your unconditional help! Like goddess, you have powers of being omniscient and you know exactly what needs to be done under a given circumstances, however grave that might sound. I am grateful to everyone in the department for suggestions, advices, helps, reagents, and laughs. vi Vishal Kumar (Zoology) and Faizule Hasan Lincoln (Biochemistry), thanks for all the jokes, foods, and movie talks. I will miss you guys. I want to thank all the baristas and people at Kofenya (for I have spent half of my grad life there) for welcoming me with open arms and serving me with much needed caffeine and laughter. Following people need special mention apart from caffeine atmosphere: Derek Reeverts (for deep conversation and advice on writing, myths, psychology, and personal matters), Joel Griggs (for sharing music and talks about pop culture), Brady Turner (Grant Morrison Fandom), Kathryn Marsman (telling me to read: The Lion, the Witch and the Wardrobe, apart from being so graceful), Bethany Bateman and Nathan Holstein (you are synonymous with great pancakes and robust coffee), Spencer Birchfield (for all those conversations, breakfasts, and jokes), Liz Snyder (Nothing would have been possible without you setting up this great atmosphere), Erin Sams, Sevika Balachandra, and Cecilia Stelzer (for reading and commenting on my stories). All of you will stay in my heart and in my memories. All of you will probably appear in my writings in one form or another. For keeping me sane throughout PhD, I owe a great deal to my friends: Yadvinder Singh (for all those philosophical discussion about Mahabharata and life), Yogi Singh Rajawat (for telling me things I should be doing like a big brother), Sandeep Kumar (just thinking about you makes me happy), Ram Prakash Kamal (for understanding me), Amandeep Dhaliwal (for laughing at my jokes), Kavita (for constantly asking about my wellbeing) and Karuna (for reminding me about my roots: Haryana). My dear family for your love and support in everything I wanted to do in life. Mummy and Papa for all the sacrifices you have made for your children and making me who I am. Arti and Sarita, my lovely sisters, for loving, fighting, and arguing with me, sharing details about the adventures for growing Arpita, Riya, and Chetan on weekly basis. Chand Singh and Dheeraj Chauhan, my elder and younger brothers, former for teaching me things, I probably don’t have memories of, and latter for being my quintessential ‘filmy’ younger brother who thinks world should understand him. Roshan Singh and Surinder Kumar, my brother-in-laws, for all the happiness you brought to me and my family. Satbir Kajal, my brother, for being there for me every single time and my buwa for her unconditional affection. Thank you everyone. vii General Introduction Viruses encounter many challenges inside the host cell that impact their survival and successful progeny production.