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Key Virus-Host Interactions Required for Arenavirus Particle Assembly and Release Christopher Michael Ziegler University of Vermont

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University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2017 Key Virus-Host Interactions Required For Arenavirus Particle Assembly And Release Christopher Michael Ziegler University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Cell Biology Commons, and the Virology Commons Recommended Citation Ziegler, Christopher Michael, "Key Virus-Host Interactions Required For Arenavirus Particle Assembly And Release" (2017). Graduate College Dissertations and Theses. 755. https://scholarworks.uvm.edu/graddis/755 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. KEY VIRUS-HOST INTERACTIONS REQUIRED FOR ARENAVIRUS PARTICLE ASSEMBLY AND RELEASE A Dissertation Presented by Christopher Michael Ziegler to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Cellular, Molecular and Biomedical Sciences May, 2017 Defense Date: March 24th, 2017 Dissertation Examination Committee: Jason W. Botten, Ph.D., Advisor Bryan A. Ballif, Ph.D., Chairperson Christopher D. Huston, Ph.D. Dimitry Krementsov, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Viruses are infectious agents that must infect the cells of living organisms in order to reproduce. They have relatively simple genomes which encode few proteins but can compensate for their simplicity by hijacking components of their cellular hosts. Arenaviruses, a family of zoonotic viruses carried by rodents, encode only 4 proteins. One of these proteins, Z, is responsible for several functions during the virus life cycle including driving the formation and release of new virus particles at the plasma membrane of infected cells. Relatively little is known about how this viral protein is regulated or the complement of host proteins it engages in order to produce new virus particles or augment Z’s other functions. To address this gap in knowledge, mass spectrometry was used to identify phosphorylation sites in the Old World arenavirus, lymphocytic choriomeningitis virus (LCMV) Z protein. Phosphorylation sites were identified at serine 41 (S41) and tyrosine 88 (Y88). Functional studies using recombinant (r)LCMV containing mutations at these phosphorylation sites revealed that both were important for the production of defective interfering (DI) particles. DI particles are replication-incompetent virus particles that interfere with the production of infectious virus and mitigate its cytopathic effect. While a mutation that mimics phosphorylation at S41 reduced LCMV’s ability to produce both infectious and DI particles, this mutation had a much stronger impact on DI particles. Production of DI particles in Y88-mutant rLCMV was drastically reduced while the impact on infectious virus was minimal. Y88 lies within a type of viral late domain (PPXY) also found in matrix proteins of several disparate virus families where it has been shown to drive infectious virus release by recruiting the membrane scission machinery of the cellular endosomal sorting complex required for transport (ESCRT). Inhibition of the ESCRT pathway drastically reduced LCMV DI particle but not infectious virus release indicating that Z’s PPXY late domain and the cellular ESCRT complex are required specifically for the production of DI particles. Mass spectrometry was also used to identify host protein partners of Z as well as the host proteins recruited into virus particles for the New World arenavirus, Junín (JUNV). ESCRT complex proteins were enriched in JUNV virus-like particles (VLPs) and bona fide virions. In contrast to LCMV, inhibition of the ESCRT complex resulted in significantly less infectious JUNV release. This indicates that the ultimate role of ESCRT engagement by the Old World arenavirus, LCMV, differs from that of New World, JUNV. This work represents the first demonstration that a viral protein motif and the host machinery it engages selectively drive DI particle production independently of infectious virus. It also suggests that host cell kinases can dynamically regulate the production of DI particles through phosphorylation of Z. Finally, the late domain-mutant rLCMV generated in these studies represents the first LCMV strain known to produce undetectable levels of DI particles which provides the opportunity to assess the impact that a loss of DI particles has on the ability of LCMV to establish or maintain a persistent infection. CITATIONS Material from this dissertation has been published in the following form: Ziegler, C.M., Eisenhauer, P., Bruce, E.A., Weir, M.E., King, B.R., Klaus, J.P., Krementsov, D.N., Shirley, D.J., Ballif, B.A., Botten, J.. (2016). The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles. PLoS Pathogens, 12:e1005501. Ziegler, C.M., Eisenhauer, P., Bruce, E.A., Beganovic, V., King, B.R., Weir, M.E., Ballif, B.A., Botten, J.. (2016). A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles. Journal of General Virology, doi:doi:10.1099/jgv.0.000550. ii ACKNOWLEDGEMENTS I would like to thank my advisor, Jason Botten, for giving me this opportunity and for being such a positive and professional role model. It has been great to work with a fellow Minnesotan and I have really enjoyed my time in your lab. I would like to thank my colleagues in the Botten lab for all the support and advice over the years and for making the lab such a positive and enjoyable place to work. I would especially like to thank Phil Eisenhauer for all of the hours he put in to support this work. I have had the pleasure of working with several undergraduate students over the last few years including Vedran Beganovic, Loan Dang, and Jamie Kelly. I would like to thank them for all of their hard work and recognize their significant contributions to these projects. I would also like to thank them for giving me the opportunity to guide them through their research projects. Lastly, I would like to thank Terri Messier and Barry Finette for giving me a start in science. Terri took me under her wing when I was a young student and I will not forget the impact that she has had in getting me to where I am today. I would like to also thank my friends and family who have provided the support that has allowed me to reach this point. My parents, who imparted on me a work ethic that has driven me to achievement in many aspects of my life, but while also teaching me to stop and smell the roses once in a while. My two brothers, Matt and Jesse, who have always kept me motivated and provided support in many ways when I needed iii it. My older brother Jesse has been a role model for me in more ways than he probably realizes throughout my life. And finally, I would like to thank my girlfriend and best friend Catherine for all the support and encouragement through this endeavor. It wouldn’t have been nearly as great without you in my life. iv TABLE OF CONTENTS Page CITATIONS ....................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ......................................... 1 1.1. Introduction .............................................................................................................. 1 1.2. Arenaviruses ............................................................................................................ 3 1.3. The Z Matrix Protein ............................................................................................... 8 1.4. Defective Interfering Particles ............................................................................... 14 1.5. The ESCRT Pathway and Late Domains ............................................................... 21 1.6. Summary ................................................................................................................ 30 References ..................................................................................................................... 32 CHAPTER 2: THE LYMPHOCYTIC CHORIOMENINGITIS VIRUS MATRIX PROTEIN PPXY LATE DOMAIN DRIVES THE PRODUCTION OF DEFECTIVE INTERFERING PARTICLES .......................................................................................... 52 Abstract ......................................................................................................................... 53 Author Summary ........................................................................................................... 54 Introduction ................................................................................................................... 55 Results ..........................................................................................................................
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    The Levels of Retinoic Acid-Inducible Gene I Are Regulated by Heat Shock Protein 90- α Tomoh Matsumiya, Tadaatsu Imaizumi, Hidemi Yoshida, Kei Satoh, Matthew K. Topham and Diana M. Stafforini This information is current as of October 2, 2021. J Immunol 2009; 182:2717-2725; ; doi: 10.4049/jimmunol.0802933 http://www.jimmunol.org/content/182/5/2717 Downloaded from References This article cites 44 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/182/5/2717.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Levels of Retinoic Acid-Inducible Gene I Are Regulated by Heat Shock Protein 90-␣1 Tomoh Matsumiya,*‡ Tadaatsu Imaizumi,‡ Hidemi Yoshida,‡ Kei Satoh,‡ Matthew K. Topham,*† and Diana M. Stafforini2*† Retinoic acid-inducible gene I (RIG-I) is an intracellular pattern recognition receptor that plays important roles during innate immune responses to viral dsRNAs.