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Structure-Function Relationships of the Nucleocapsid in Vesicular Stomatitis Virus Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 12-10-2018 Structure-Function Relationships of the Nucleocapsid in Vesicular Stomatitis Virus Ryan Gumpper Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Gumpper, Ryan, "Structure-Function Relationships of the Nucleocapsid in Vesicular Stomatitis Virus." Dissertation, Georgia State University, 2018. https://scholarworks.gsu.edu/chemistry_diss/153 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. STRUCTURE-FUNCTION RELATIONSHIPS OF THE NUCLEOCAPSID IN VESICULAR STOMATITIS VIRUS by RYAN GUMPPER Under the Direction of Ming Luo, PhD ABSTRACT Understanding the basic structure-function relationships that guide the biology of various pathogenic viruses is the cornerstone in the development of antiviral therapies. One group of highly pathogenic viruses, which desperately needs novel antiviral therapies, are Negative Strand RNA viruses. This group of viruses contains some of the most highly pathogenic and pandemic viruses known: like Ebola, Influenza, Marburg, and Rabies viruses. Between 2013 and 2016, during the latest endemic outbreak of Ebola, the World Health Organization estimated that it caused over 11,000 deaths. However, without in-depth understanding of the basic science that guides the replication of these viruses, design of targeted therapies is hard. Utilizing the model virus for Negative Strand RNA viruses, Vesicular Stomatitis virus, this work studies the structure-function relationships of the nucleocapsid protein. Since the nucleocapsid encapsulates the virus’ genomic material this protein is increasingly important study. Furthermore, the virus cannot undergo replication if the polymerase does not recognize the nucleocapsid for viral RNA synthesis. This work takes a three-pronged approach when investigating the nucleocapsid: development of a novel antiviral to target the nucleocapsid, the use of structural information to study the possible interactions between the nucleocapsid and the polymerase, and a study of the genomic constraints placed on polymerase activity by the nucleocapsid. Utilizing an already characterized class of compounds, polyamides, we showed that it is possible to target the genomic RNA in the nucleocapsid of a Negative Strand RNA virus and inhibit viral replication. This is the first known instance of this occurring in Negative Strand RNA viruses. To examine the structural interactions between the nucleocapsid and the polymerase a flexible loop was hypothesized to be integral toward the formation of an active polymerase complex. Alanine scanning deletions yielded an abolishment in polymerase activity, which could be restored after compensatory mutations were allowed to occur. Finally, the nucleotide content sequestered within the nucleocapsid has a direct effect on the processivity of the polymerase complex, and ushers in a paradigm changing model for how one examines Negative Strand RNA virus activity and the evolutionary constraints placed on the genomic RNA. Merging the findings of these studies together, understanding the structure-function relationships of the nucleocapsid can yield a lot of knowledge about the viral life-cycle of the entire Negative Stranded RNA virus family. INDEX WORDS: Negative Strand RNA virus, Nucleocapsid, Antiviral therapies, Vesicular Stomatitis Virus STRUCTURE-FUNCTION RELATIONSHIPS OF THE NUCLEOCAPSID IN VESICULAR STOMATITIS VIRUS by RYAN GUMPPER A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2018 Copyright by Ryan Gumpper 2018 STRUCTURE-FUNCTION RELATIONSHIPS OF THE NUCLEOCAPSID IN VESICULAR STOMATITIS VIRUS by RYAN GUMPPER Committee Chair: Ming Luo Committee Members: Jenny Yang Gregory Poon Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University November 2018 iv DEDICATION I would like to dedicate this dissertation to my Mom, Dad, Brother, and fiancé, Suela. Without the love and support of all of you, none of this would be possible. v ACKNOWLEDGEMENTS First, I would like to acknowledge my advisor, Dr. Ming Luo, for always leading by example and teaching me what it means to be a true scientist. I would like to thank him especially for taking me into his lab when I thought my entire PhD was in jeopardy and giving me the chance to succeed. Furthermore, I would also like to thank him for the ongoing moral and academic support through this process and allowing me the space to explore new ideas not only scientifically, but also personally. Without his help, none of this would have been possible. I would also like to thank both of my committee members Dr. Gregory Poon and Dr. Jenny Yang. To Dr. Yang whom I have interacted with since my first day in graduate school in her Protein Structure and Function class. From being on my oral exam committee, to putting together the wonderful joint group meetings, and who has offered nothing but amazing scientific and professional advice all the way through my graduate career. Also, to Dr. Gregory Poon who has offered a plethora of moral support and has offered many insights into the ins-and-outs behind science and the scientific process. I always know that I can come to you for your sage advice. I would also like to thank Dr. Jiafeng Geng and Dr. Kednerlin Dornevil, both members of my former lab. Not only did both of you teach me all of the necessary laboratory techniques but were also an invaluable part of my growth as a scientist when I first started graduate school. As a freshmen graduate student, having senior members take you under their wing, like both of you did, did more to shape my career than you can imagine. Also, to the current members of my lab, especially Weike (Eric) Li and Ross Terrell. To all the inspiring conversations, both scientific vi and non-scientific while toiling away for the endless hours in lab. Both of your friendships mean the world to me and helped keep me sane when times were tough. I would also like to thank my family: my Mom, Dad, and Brother. Being far apart these past 5 years has been tough, but without your constant love and support throughout this entire process I surely wouldn’t have been able to do it. All of you have raised me to be an independent thinker and a hard worker, and without these traits which have been so ingrained into the fabric of my being I would not be where I am today. Thank you for traveling all those times to see me, as graduate school gets very busy at times, and I am eternally grateful for all your sacrifices in time, money, and heartache at being far away. Sometimes achieving something worth doing takes a lot of sacrifice but know that my love for you all is unconditional, and I look forward to the days we can spend more time together. Without you guys as my foundation, I wouldn’t have been able to build the tower that you see today. I love you guys and look forward to the future! Finally, I would like to especially thank my partner in crime, the love of my life, and other-half, my fiancé Suela. To this day, your love and devotion continues to inspire me to reach to new heights. Without you by my side every step of the way life would not be as happy, fulfilling, or fun. Growing up together through graduate school will be a time in our lives that we will always cherish. It was during this time that our relationship blossomed, and our love grew to enormities that I could never comprehend to exist. Through all the failed experiments, the late nights and early mornings in the lab, and plenty of tears you have been my rock. Every day with you is filled with adventure and love, and without your emotional and professional support I would not be half the man that I am today. This journey has not been easy, and the road has not been straight, but I cannot think of any other person I would rather have by my side than you. vii Thank you for being you and I will continue to love you as this next exciting step in our lives begins. I love you, and I always will—here’s to the next adventure. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ V LIST OF TABLES ....................................................................................................... XIII LIST OF FIGURES ..................................................................................................... XIV LIST OF ABBREVIATIONS ..................................................................................... XVI 1 GENERAL INTRODUCTION: WHY STUDY VIRUSES? .................................. 1 1.1 General History of Virology ............................................................................... 1 1.2 Viral Evolution, Classification System, and Antiviral Therapies ................... 3 1.2.1 Viral Evolution ................................................................................................ 4 1.2.2 Viral Classification System ............................................................................. 7 1.2.3 Antiviral Therapies ......................................................................................
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