AN ABSTRACT OF THE DISSERTATION OF Nathan E. Jespersen for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on September 10, 2019. Title: Viral Targeting of Host Hubs: Interactions between the Rabies Virus and LC8 Abstract approved: ______________________________________________________ Elisar J. Barbar As obligate intracellular parasites, viruses rely on host machinery for their own reproduction. Viruses are therefore required to interact with a wide variety of host proteins, despite limitations in viral genome size. The most parsimonious method of success is to hijack central and essential proteins, known as hub proteins. LC8 is a notable example of a hub protein, and has been shown to interact with more than 100 eukaryotic partners in various unrelated pathways. An increasing number of studies have noted interactions between LC8 and viruses, including the Ebola, rabies, and rotaviruses; however, LC8’s role within these systems is unclear. This thesis examines the structural and functional properties of hub proteins, with a particular focus on LC8 and its interactions with the rabies virus. Three chapters of original work include two primary research reports and one review/opinion piece. The first research report, Chapter 2, structurally characterizes the interaction between LC8 and the rabies virus phosphoprotein (RavP). We use insights gleaned from our structural studies to predict and test potential roles for LC8 in the rabies virus infection cycle, and demonstrate that LC8 is important for efficient viral polymerase activity. Chapter 3 is an in-depth study of the LC8 recognition motif, where we examine both the structural and functional plasticity of LC8, and identify and validate many new LC8 interactions. We also develop a tool that can be used to predict LC8 binding motifs in proteins of interest, which will greatly improve the ability of those outside of the LC8 field to recognize, test, and validate partner proteins. In Chapter 4 we present a variety of new ideas about what qualities describe a linear motif-binding hub protein. This work provides ideological and linguistic suggestions for important structural and functional features of hubs that underlie their plasticity. It then further describes how viruses take advantage of these features to more efficiently hijack host pathways. Finally, Chapter 5 discusses the impacts of my thesis work, and outlines some potential future projects. Each individual chapter builds on the previous one to create an expanding view of the importance of linear motif-binding hubs for infectious viruses. ©Copyright by Nathan E. Jespersen September 10, 2019 All Rights Reserved Viral Targeting of Host Hubs: Interactions between the Rabies Virus and LC8 by Nathan E. Jespersen A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented September 10, 2019 Commencement June 2020 Doctor of Philosophy dissertation of Nathan E. Jespersen presented on September 10, 2019 APPROVED: Major Professor, representing Biochemistry and Biophysics Head of the Department of Biochemistry and Biophysics Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Nathan E. Jespersen, Author ACKNOWLEDGEMENTS First and foremost, I would like to thank Dr. Elisar Barbar, for all of your insight, assistance, mentoring, and prodding over the years. I have no idea how many hours I spent chatting with you about whatever new theory was on my brain, and your enthusiasm in those situations was always so wonderful. Sarah and I used to joke about how if we were ever feeling nervous during a presentation, we could glance over at you and see you smiling and supportive, and it would perk us right up. Your willingness to let me look into a new idea allowed me to get a very broad skillset, and you always encouraged me to consider some grant or fellowship. During the few occasions where I was feeling really down about science, you were marvelously supportive. I am super fortunate to have had you as an advisor! I have had some terrific collaborators over the years, many of whom are my French compatriots, including Dr. Marc Jamin, Dr. Cedric Leyrat, and particularly Dr. Danielle Blondel and everyone else in Gif-sur-Yvette. You all opened up a whole new set of experiments to me, and were extremely patient (especially considering my very poor French). I’m not sure how my RavP work would have come together without you. The Biochemistry and Biophysics Department at OSU is great. Regardless of the professor, I was never reticent to go by their office to pester them about science. I would say the best thing about graduate school is that there is probably somebody right down the hall who knows a lot about any given subject, and our department is tight-knit enough to swing by for a chat. I would like to thank all of my committee members. Dr. Theo Dreher, your virology course was one of my favorites I have ever taken, and I think that your input during my prelims strongly impacted the way my future experiments proceeded and my success with the Chateaubriand Fellowship. My initial GCR, Dr. Sandra Loesgen, Thanks for taking the time to sit down with me and iron out some of the finer points of my fellowship application. Dr. Brian Dolan, you saved the day by agreeing to be my GCR at the last moment! Dr. Afua Nyarko – not only have you mentored me as a member of my committee, but you also taught me many of the wet-lab protocols that I still follow. Even though I’ve had to resort to trickery to learn anything about your life, I still really appreciate all of our interactions. Dr. Ryan Mehl, I’ve had so many great conversations with you about science, life, and work. I often sought you out when I was feeling particularly uncertain, and you always lent me an ear and gave me some good advice. Thanks! The Barbar Besties (otherwise known as the current Barbar lab members Aidan Estelle, Kayla Jara, and Heather Forsythe) are quite a group. I can’t say I fully understand most of the conversations that happen in the lab (What’s Aidan hating on now? Pomegranates?), but it’s always entertaining! Sanjana Saravanan, you were the best undergrad I ever had. Patrick Reardon, you don’t quite go in the Barbar lab section, but with the amount of time you spend in our lab helping us with NMR questions, I think it’s pretty appropriate. My ol’ teammate Dr. Sarah Clark, with whom I have so many great memories in the lab and outside of it. Whether it was commiserating or co-celebrating, it was always better with you! I also want to thank my family and friends (some of which are included in other categories). Mom and Dad, you guys should win awards for selfless support. I know I moved across the country and don’t get home that frequently, but you guys (almost) never complain about that, and always make sure to remind me how proud of me you are. It makes me feel nice. Amongst my friends, I’d like to give a particular shout-out to Dr. Andrew Brereton, who is one of the most interesting people to chat with I have ever met. Our graveyard of forgotten theories would be something to behold! Last but not least, my wonderful and intelligent wife Michelle. Your calm mien relaxed me when I was manic, and your dedication to sleep kept me healthy. Without your regular and on-point reminders, who knows how many things would have been left undone. CONTRIBUTION OF AUTHORS Cedric Leyrat contributed to the molecular dynamics simulations in Chapter 2. Marc Jamin, Jeam-Marie Bourhis, and Francine Gérard contributed to SAXS data collection and analysis in Chapter 2. Danielle Blondel guided in cell experiments in Chapter 2. Ylva Ivarsson and Cecilia Blikstad performed phage display experiments from Chapter 3. Anna Akhmanova and York-Christoph Ammon collected cell images shown in Chapter 3. Aidan Estelle contributed to the writing and production of the LC8Pred algorithm in Chapter 3. Sarah Clark contributed to figure design throughout my thesis. Elisar Barbar was involved in all steps of this thesis work. TABLE OF CONTENTS Page Chapter 1: Introduction…………………………………………………………………….…..1 Dynein Light Chain 1: More Than a Motor Protein Subunit………………………….2 History and Overview………………………………………………………...3 LC8 Structure………………………………………………………………...4 Intrinsic Protein Disorder…………………………………………………….5 LC8 Function………………………………………………………………....6 LC8 Feedback Regulation……………………………………………………8 The Rabies Virus…………………………………………………………………….10 History and Epidemiology…………………………………………………..10 Structure…………………………………………………………………….13 Rabies and LC8……………………………………………………………..15 Biophysical Techniques……………………………………………………………...16 Isothermal Titration Calorimetry……………………………………………16 Circular Dichroism………………………………………………………….16 Multi-Angle (Laser) Light Scattering……………………………………….17 Small-Angle X-ray Scattering………………………………………………17 Nuclear Magnetic Resonance……………………………………………….17 Dissertation Contents………………………………………………………………...18 Chapter 2: The LC8-RavP Ensemble Structure Evinces a Role for LC8 in Regulating Lyssavirus Polymerase Functionality……………………...…………………………………20 Abstract……………………………………………………………………………...21 Introduction………………………………………………………………………….22 Results……………………………………………………………………………….25 Discussion………………………………………………………………………...…39 Materials and Methods………………………………………………………………44 Acknowledgements………………………………………………………………….53 Chapter 3: Systematic Identification