Understanding Prototype Foamy Virus Integrase Site Selection, Activity, and Stability Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Randi Michelle Mackler, BS Biomedical Sciences Graduate Program The Ohio State University 2018 Dissertation Committee Dr. Kristine Yoder, PhD, Advisor Dr. Michael Freitas, PhD Dr. Jesse Kwiek, PhD Dr. Li Wu, PhD Copyrighted by Randi Michelle Mackler 2018 Abstract HIV is a worldwide pandemic that remains incurable. Recent statistics show that in the United States alone, ~15 out of every 100,000 people were newly infected with HIV-1 in one year. The barrier to a cure is a reservoir of cells with viral DNA stably integrated into their genome, yet are not killed by the immune system. The integration step of the retroviral life cycle is crucial in formation of this reservoir. Viral DNA integration is catalyzed by the protein integrase (IN). We study HIV-1 IN as well as prototype foamy virus (PFV) IN. PFV IN is used as a model for HIV-1 integration, as HIV-1 IN inhibitors also block PFV IN activity. This implies that the two proteins have similar catalytic mechanisms. However, we have found some differences between PFV IN and HIV-1 IN function. We determined that PFV IN could utilize Ca2+ for strand transfer, unlike HIV-1 IN. In addition, though HIV-1 IN has been reported to rapidly commit to its target DNA, PFV IN does not commit within an hour. Therefore, there are likely differences in searching and target capture mechanisms between the two INs. A benefit to using PFV IN is that it can be readily assembled with oligomers that mimic viral cDNA ends to form a complex termed an intasome. The PFV intasome contains a tetramer of PFV IN and two oligomer DNAs. Interestingly, we found in vitro that these intasomes aggregate at 37°C. Full-length intasomes aggregate more than those containing ii truncated PFV IN outer subunits, particularly deleting the carboxyl terminal domain (CTD). Aggregation can be prevented by using high non-physiological salt concentrations or with the addition of small molecule protocatechuic acid (PCA). This finding is useful for future experiments that require longer lifetimes of PFV intasomes. PFV intasomes with full-length or truncated outer subunits were also used to understand integration into nucleosomes. Integration into chromatin is still not well understood. Chromatin, which condenses genomic DNA to fit into a cell’s nucleus, is comprised of basic units called nucleosomes. Our goal is to understand how IN chooses its site when integrating into nucleosomes. We altered either nucleosomes or PFV IN to understand how changes impact integration activity and site specificity. Perturbations include using nucleosomes with specific histone posttranslational modifications (PTMs), altering salt concentration, and utilizing PFV IN truncation mutants. We hypothesize that major grooves with highly bent DNA that are not occluded by histone proteins are the most favored sites for integration. However, our studies showed that not every distorted and exposed major groove is favorable for integration, suggesting that there is more to PFV IN site selection. Interestingly, deleting the CTDs of the outer subunits of the IN tetramer greatly increases integration efficiency to linear DNA, but this effect is largely lost with integration into nucleosomes. These truncated mutant complexes are the most impacted by increasing salt concentrations, and affinity purification experiments showed that the PFV IN ΔCTD intasome interacts weakly with nucleosomes compared to wild type. Thus, our data supports the hypothesis that the CTDs of PFV IN directly bind to nucleosomes. In particular, the interaction is at least partially mediated by the histone tails. Results of these iii studies will expand the knowledge in the field and will be crucial to development of novel therapeutics to combat HIV-1 at the integration step. This work can also inform design of novel retroviral gene therapy vectors. iv Dedication To Nick for loving me and taking care of me physically and emotionally. To my parents for their love, support, and advice through all of my ups and downs. Without their support, I would not be where I am today. v Acknowledgments I am very appreciative of my support system of family, friends, peers, and mentors. To my advisor Dr. Kristine Yoder, and committee members Drs. Jesse Kwiek, Michael Freitas, and Li Wu – thank you for helping me think deeply about my science and teaching me to think critically. Thank you program directors Drs. Joanna Groden and Jeffrey Parvin for guiding me through my graduate career and making sure I am prepared for my future. I would also like to acknowledge Drs. Richard Fishel and John Gunn for being additional mentors to me. Thank you to my colleagues in the Yoder and Fishel labs for the friendship, advice, and mentorship. I would particularly like to thank Miguel Lopez and Dr. Gayan Senavirathne for their endless support and patience, long brainstorming sessions and making a great lab environment. Thank you to everyone in the Center for Retrovirus Research for their insightful questions and ideas and making a great community of retrovirologists at OSU. I would also like to thank the scientists who have helped me get to graduate school to begin with. Thank you Dr. William Jacobs for truly sparking my interest in science research and Dr. Oren Mayer for making sure I had a great foundation of pipetting, aseptic technique, and experimentation. Thank you to Drs. William Coleman and Ashley Rivenbark for guiding me through graduate school applications and being great mentors. vi I would like to acknowledge my family and friends who have stood by me as I have gone on this crazy journey of graduate school. Christina – who knew meeting at another graduate school interview we would become best friends? We have been through it all together and I don’t think I would have made it through without you as my constant support, soundboard, and cheerleader. To my sister Hayley – you were the first one to say I would grow up to be a scientist and look where I am now! I have always been appreciative of your support. To my fiancé Nick – you really deserve an honorary PhD for how much you have supported me throughout this process; I love you very much. Lastly, I would like to thank my incredible parents Karen and Mark Mackler. You have always believed in me when I didn’t believe in myself and have been a constant source of love and wisdom. I could not have done this without you. vii Vita 2009…………………………………………John F. Kennedy High School, NY 2013………………………………………....B.S. Chemistry – Biochemistry Track, University of North Carolina at Chapel Hill, NC 2013 – Present………………………………Ph.D. Candidate, Biomedical Sciences, The Ohio State University, OH Publications Mackler RM, Jones ND, Gardner AM, Senavirathne GS, Lopez MA Jr, Altman MP, Fishel R, Yoder KE. Prototype foamy virus integrase carboxyl terminal domains dictate site selection into nucleosomes. J Biol Chem. In preparation. Mackler RM, Jones ND, Baltierra Jasso LE, Messer RK, Yoder KE. Retroviral integrase drug resistant mutant displays a novel mechanism of reduced viral fitness. Virology. In preparation. Jones ND*, Mackler RM*, Lopez MA Jr*, Baltierra Jasso L, Altman MP, Senavirathne GS, Yoder KE. Prototype foamy virus intasome aggregation is mediated by outer protein viii domains and prevented by protocatechuic acid. Sci Rep. Accepted upon minor revisions. * Indicates equal contribution Mackler RM, Jones ND, Lopez MA Jr, Howard CJ, Fishel R, Yoder KE. Nucleosome DNA unwrapping does not affect prototype foamy virus integration efficiency or site selection. PLOS ONE. Under Revision. Mackler RM, Lopez MA, Osterhage MJ, Yoder KE. Prototype foamy virus integrase is promiscuous for target choice. Biochem Biophys Res Commun. 2018 Sep 10;503(3):1241- 1246. PMID: 30017200; PMCID: PMC6119477. Mackler RM, Lopez MA Jr, Yoder KE. Assembly and Purification of Prototype Foamy Virus Intasomes. J Vis Exp. 2018 Mar 19;(133). PMID: 29608167; PMCID: PMC5933227. Lopez MA Jr, Mackler RM, Altman MP, Yoder KE. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J Vis Exp. 2017 Dec 8;(130). PMID: 29286489; PMCID: PMC5755535. Lopez MA Jr, Mackler RM, Yoder KE. Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase. J Virol Methods. 2016 Sep;235:134-138. PMID: 27269588; PMCID: PMC4992616. Field of Study Major Field: Biomedical Sciences Graduate Program Emphasis: Microbial Pathogenesis (Virology) ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv Chapter 1. Introduction ......................................................................................................