Repurposing the Human Immunodeficiency Virus (Hiv) Integrase
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REPURPOSING THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) INTEGRASE INHIBITOR RALTEGRAVIR FOR THE TREATMENT OF FELID ALPHAHERPESVIRUS 1 (FHV-1) OCULAR INFECTION A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Matthew Robert Pennington August 2018 © 2018 Matthew Robert Pennington REPURPOSING THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) INTEGRASE INHIBITOR RALTEGRAVIR FOR THE TREATMENT OF FELID ALPHAHERPESVIRUS 1 (FHV-1) OCULAR INFECTION Matthew Robert Pennington, Ph.D. Cornell University 2018 Herpesviruses infect many species, inducing a wide range of diseases. Herpesvirus- induced ocular disease, which may lead to blindness, commonly occurs in humans, dogs, and cats, and is caused by human alphaherpesvirus 1 (HHV-1), canid alphaherpesvirus (CHV-1), and felid alphaherpesvirus 1 (FHV-1), respectively. Rapid and effective antiviral therapy is of the utmost importance to control infection in order to preserve the vision of infected people or animals. However, current treatment options are suboptimal, in large part due to the difficulty and cost of de novo drug development and the lack of effective models to bridge work in in vitro cell cultures and in vivo. Repurposing currently approved drugs for viral infections is one strategy to more rapidly identify new therapeutics. Furthermore, studying ocular herpesviruses in cats is of particular importance, as this condition is a frequent disease manifestation in these animals and FHV-1 infection of the cat is increasingly being recognized as a valuable natural- host model of herpesvirus-induced ocular infection First, the current models to study ocular herpesvirus infections were reviewed. Next, the efficacy of raltegravir was evaluated using a novel corneal explant model. Raltegravir is a human immunodeficiency virus (HIV) integrase inhibitor that was recently shown to poses antiviral activity against human herpesviruses. Then, electric cell-substrate impedance sensing (ECIS) was evaluated as a novel methodology to study the replication kinetics of herpesviruses. It was also used to characterize a fluorescently labeled FHV-1, created using CRISPR/Cas9 genome. Next, it was found that raltegravir inhibits both viral DNA synthesis initiation and late gene expression, a mechanism consistent with inhibition of viral ICP8. Finally, RNA sequencing was used to explore the indirect effects of raltegravir on the host. It was found that raltegravir treatment promoted the expression of anti-angiogenic factors and altered the metabolism of the host cells, both of which may be beneficial therapeutically. These results, combined with a recent in vivo study in experimentally infected cats, demonstrates that raltegravir is a viable treatment option for FHV-1 and warrants further investigations into its clinical potential against other herpesviruses. BIOGRAPHICAL SKETCH Matthew Pennington was born in Shawnee, KS, where he grew up. He first attended St. Joseph’s Catholic School and Good Shepherd Catholic School for grade school and then Rockhurst High School (Kansas City, MO). Matthew pursued his baccalaureate degree at Seton Hall University in South Orange, NJ. At Seton Hall, Matthew majored in Biological Sciences, minored in Chemistry, and participated in the both the Biological Sciences and the University Honors Programs. While there, he began research as a freshman studying the effects of the P- TEFb inhibitor flavopiridol on human gammaherpesvirus 8 transcription and replication, leading to his interest in antiviral drug development. During his undergraduate studies, Matthew studied adolescent elephant behavior at the Pongal Game Reserve (KwaZulu-Natal, South Africa), their ecological impacts on their habitat, and methods of population management for his senior theses, driving his interest in animal biology. He graduated in 2013, Summa cum laude, and was awarded the Biology Department Honors Citation by the College of Arts and Sciences and the Charles F. and Hannah Chelius Stark Prize by the University Honors Program Faculty. Matthew then enrolled at Cornell University as a graduate student in Immunology and Infectious Diseases to pursue is interest in study the pathology and treatment of animal viruses. He joined the lab of Dr. Gerlinde Van de Walle in the summer of 2014 to study animal herpesviruses, including felid alphaherpesvirus 1 and equid gammaherpesviruses 2 and 5. Following completion of his graduate studies, Matthew accepted a Post-Doctoral Fellowship at Massachusetts Eye and Ear Infirmary (Massachusetts General Hospital, Harvard Medical School) in the lab of Dr. James Chodosh. There he will study the immunopathogenesis of human ocular adenovirus infection. iii ACKNOWLEDGEMENTS There are many people who have helped me throughout my time at Cornell University and without whom I would not have been able to complete this degree. First and foremost, I would like to thank my mentor, Dr. Gerlinde Van de Walle, who allowed me to explore and to take this research in the direction I found interesting, as well as providing the opportunities to dive into other, unrelated, projects. She has provided me with unwavering support over the years and has always been there to provide help and advice. I also owe a special thanks to my special committee, Drs. Colin Parrish, Bettina Wagner, and Eric Ledbetter, for their support, collaborations, enthusiasm, and useful insights to my work. All of the members of our lab, both past and present, including Melissa Ledet, Becky Harman, Gat Rauner, Joy Tomlinson, Lauren Tofano, Mike Fort, and other members, visiting researchers, and undergraduate students, have made this time an enjoyable and intellectually stimulating experience. In particular, I have to thank Becky for always happily answering my myriad of questions on everything from help with western blots and flow cytometry to opinions on where I should go hiking. Another special thanks is due to Heather Callaway for her help with protein expression, revising papers and grants, random side projects and ideas that never led anywhere, and more. Thanks to Jen Grenier of the Cornell University RNAseq core for patiently teaching me the basics of RNA sequencing and helping me to analyze and reanalyze my data. Additionally, thanks to Don Millar for help designing cloning constructs, Ashley Roloson and Liberty Research for tissue collection, and the staff of Applied BioPhysics for their assistance optimizing the ECIS assays and analyzing the data. Additionally, I owe a debt of gratitude to the Cornell University Feline Health Center for its continued funding of me and our studies over the duration of my Ph.D. iv Lastly, I would like to thank all of my friends and family for their support on this journey. I have enjoyed our hikes, dog play-dates, brewery and winery trips, knitting nights, and more immensely. You all have made my time at Cornell more enjoyable and rewarding. I will always be grateful to these friends and future colleagues for their help and support during my time at Cornell University. v TABLE OF CONTENTS BIOGRAPHICAL SKETCH iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS vi LIST OF FIGURES x LIST OF TABLES xiii LIST OF ABBREVIATIONS xiv CHAPTER ONE: AN INTRODUCTION TO OCULAR HERPESVIRUS INFECTION AND THE CHALLENGES OF ANTIVIRAL DRUG DEVELOPMENT 1.1. Herpesvirus-associated ocular disease 2 1.2. Current vaccines and therapies for human and animal patients with ocular herpes 7 1.2.1. Humans 7 1.2.2. Dogs 8 1.2.3. Cats 9 1.3. Strategies to accelerate antiviral drug development 11 1.4. Repurposing raltegravir as a therapy for ocular herpesvirus infection 15 1.5. References 17 CHAPTER TWO: NEW PARADIGMS FOR THE STUDY OF OCULAR ALPHAHERPESVIRUS INFECTIONS: INSIGHTS INTO THE USE OF NON- TRADITIONAL HOST MODEL SYSTEMS 2.1. Summary 26 2.2. Introduction 27 2.3. In vitro 2D cell culture systems 30 2.3.1. Immortalized cell lines 30 2.3.2. Primary corneal epithelial cells 31 2.3.3. Limitations of 2D cell culture systems 33 2.4. In vitro 3D cell culture/explant systems 34 vi 2.4.1. Corneal facsimiles 34 2.4.2. Explants 35 2.4.3. Limitations of 3D cell culture/explant systems 39 2.5. In vivo systems 41 2.5.1. Mice 41 2.5.2. Rabbits 42 2.5.3. Dogs 43 2.5.4. Cats 49 2.5.5. Limitations of non-traditional in vivo models 51 2.6. Conclusions and future prospects 53 2.7. References 55 CHAPTER THREE: A NOVEL CORNEA EXPLANT MODEL SYSTEM TO EVALUATE ANTIVIRALS AGAINST FELID ALPHAHERPESVIRUS TYPE 1 (FHV-1) 3.1. Summary 75 3.2. Introduction 76 3.3. Results 78 3.4. Discussion 89 3.5. Methods 94 3.6. References 100 CHAPTER FOUR: ELECTRIC CELL-SUBSTRATE IMPEDANCE SENSING (ECIS) TO MONITOR VIRAL GROWTH AND STUDY CELLULAR RESPONSES TO INFECTION WITH ALPHAHERPESVIRUSES IN REAL-TIME 4.1. Summary 105 4.2. Introduction 107 4.3. Results 109 4.4. Discussion 120 4.5. Methods 123 4.6. References 130 vii CHAPTER FIVE: THE HIV INTEGRASE INHIBITOR RALTEGRAVIR INHIBITS FELID ALPHAHERPESVIRUS 1 (FHV-1) REPLICATION BY TARGETING BOTH DNA REPLICATION AND LATE GENE EXPRESSION 5.1. Summary 135 5.2. Introduction 137 5.3. Results 139 5.4. Discussion 153 5.5. Methods 157 5.6. References 167 CHAPTER SIX: TRANSCRIPTOME PROFILING OF ALPHAHERPESVIRUS- INFECTED CELLS TREATED WITH THE HIV-INTEGRASE INHIBITOR RALTEGRAVIR REVEALS PRFOUND AND SPECIFIC ALTERATIONS IN HOST TRANSCRIPTION 6.1. Summary 176 6.2. Introduction 177 6.3. Results 179 6.4. Discussion 197 6.5. Methods 202 6.6. References 212 CHAPTER SEVEN: DISCUSSION: SUMMARY AND IMPLICATIONS FOR FUTURE ANTIVIRAL DEVELOPMENT 7.1. Summary of Findings 220 7.2. Prospects for raltegravir as a therapy for herpesvirus infections 221 7.2.1. Evaluation of raltegravir in vivo 221 7.2.2. Comparison of raltegravir to current FHV-1 therapies 223 7.2.3. Considerations for the practical implementation of oral and topical raltegravir 225 therapy 7.2.4. Intellectual property and cost considerations 226 7.3.