Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State, a Tool to Study Protein Triggering DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Supranee Chaiwatpongsakorn The Comparative and Veterinary Medicine Graduate Program The Ohio State University 2011 Dissertation Committee: Mark Peeples Michael Oglesbee Stefan Niewiesk Jianrong Li Copyright by Supranee Chaiwatpongsakorn 2011 ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, Pneumovirinae subfamily is the most significant respiratory pathogen in infants and second only to influenza virus in the elderly. Despite extensive efforts, no vaccines or small molecule antiviral drugs are available. The RSV fusion (F) glycoprotein has been a major target for vaccine and antiviral drug development because of its importance in the viral replication cycle, its conserved sequence and structure, its exposed position in the virion, and its strong immunogenicity. Like other paramyxoviruses, the RSV F protein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. However, the Pneumovirinae F protein is unique in that it, alone, is sufficient to mediate membrane fusion and virus infection. It is, therefore, the simplest F protein to study. It likely has the ability to attach to target cells from which position it is triggered. Neither the trigger site on the F protein nor the triggering molecule/event has been identified. To begin to study the triggering mechanism of the RSV F protein biochemically, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains ii with a 6His tag. This sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopy, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni2+ column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hatpin” shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, confirming that association is via the fusion peptide. Although it is not clear whether reduction in molarity can serve as a physiological trigger of the intact F protein during the natural infection of RSV, our study has revealed a novel, surrogate method for triggering a viral fusion protein. The availability of pretriggered RSV sF protein capable of being triggered and transformation into its posttriggered conformation enables studies of its mechanism of attachment, triggering, and refolding, a protein vaccine for adults, assays to quantify antibodies against F, discovery of the mechanism of action of drugs known to target F, and high throughput screens to identify new and better drugs against F. iii DEDICATION This dissertation is dedicated to my family and the loved ones. The first one to thank was my dad, for being a big inspiration and a role model of my life. His unequivocal support and encouragement always made me feel confident to overcome every difficulty I encountered. He always let me know that he was so proud of me, which motivates me to work harder and do my best. Thanks again for believing in me. Even though you are not here today, I always know how happy you would be to see my success. And you should know that you are a big part of it, always and will be. For my mom, this dissertation cannot be done and I would not have come this far without your unconditional love and support. You are truly my best friend who lives just a phone call away. This work is also dedicated to my brother, P‟Kob, who always understands and shares the feeling I have had from study and research when no one else could. I also dedicate this dissertation to everyone in Chaiwatpongsakorn family for their loving support. I thank them for being there to help out and comfort my parents when I and my brother were so far away in another country pursuing our doctoral degrees. Lastly, I also want to dedicate this work to P‟Jump for a wonderful time we have had together. Thanks for cheering me up, for trying to understand the person I am, and for believing in me when I lost faith in myself. iv ACKNOWLDEGEMENTS First of all, I would like to thank my advisor, Dr. Mark E. Peeples for his important support throughout this work. His wide knowledge and logical way of thinking have been a great value to me since the first day I joined the lab. I have learned a lot from him and realized for all these years how fortunate I am to have him as my mentor. In the last phase of my dissertation, he helped me to correct grammar mistakes and suggested possible improvements. Without his help I could not have finished my dissertation successfully. I am also thankful to him for allowing me to go back home several times in the past few months to take care of my dad and to perform a religious ceremony after he passed away. Thank you so much again for your generous understanding during this difficult time. I also thank Dr. Sunee Techaarpornkul for giving me a great opportunity and recommending me to Dr. Peeples. Laboratory training from her and Dr. Nusara Piyapolrungroj in cell culture and molecular biology gave me tools that turned out to be essential in my PhD research. I own a sincere gratitude to both of them. I would like to thank Drs. Richard and Raquel Epand for teaching me how to make and use liposomes and their valuable discussions and helpful v comments on my project. I am also thankful for their warm hospitality and financial support during my training in Canada. I wish to thank all past and present members of the Peeples lab for their technical support, scientific guidance, and great time we shared. It was my pleasure to share doctoral studies and life with wonderful people like Steve, Anna, Olga, and Heather. Especially, Heather, I really appreciate your assistance on my F model project, thank you so much again for your kind friendship, for sharing the glory and sadness of our grant submission deadlines and day-to-day research. I also would like to express my gratitude to my committee members, Dr. Michael Oglesbee, Dr. Stefan Niewiesk, Dr. Jianrong Li, and Dr. Joan Durbin for providing me their valuable suggestions throughout my graduate study. I would also like to extend my appreciation to many colleges from the Veterinary Biosciences graduate program and Center for Vaccines and Immunity who have assisted me in my studies and project for all these years. And for Dr. Will Ray, I thank him for his amazing work on the RSV F computational models and for allowing me to present his work in my dissertation. vi VITA 1998…………………………………………….The demonstration school of Silpakorn University, Thailand 2003…………………………………………….B. Pharmacy, Silpakorn University, Thailand (first class honor) 2004 to present ……………………………….Graduate Research Associate, Department of Veterinary Biosciences, The Ohio State University PUBLICATIONS Chaiwatpongsakorn, S., et al., Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer. J Virol, 2011. 85:3968-3977. FIELDS OF STUDY Major Field: The Comparative and Veterinary Medicine Graduate Program Minor Fields: Molecular Virology vii TABLE OF CONTENTS Abstract….. ............................................................................................................ ii Dedication ............................................................................................................ iv Acknowledgements .............................................................................................. v Vita…….... ........................................................................................................... vii List of Figures ....................................................................................................... xi Abbreviations ...................................................................................................... xiv Chapter 1: Respiratory Syncytial Virus ................................................................. 1 Introduction ........................................................................................................... 1 Taxonomy and classification................................................................................. 2 RSV organization and morphology ....................................................................... 3 The RSV life cycle ................................................................................................ 4 Attachment and entry ........................................................................... 4 Transcription, translation, and replication ............................................