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Regulation of Mrna Translation by Human Cytomegalovirus Ptrs1

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Regulation of Mrna Translation by Human Cytomegalovirus Ptrs1 REGULATION OF MRNA TRANSLATION BY HUMAN CYTOMEGALOVIRUS PTRS1 Heather Ashley Vincent A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology in the School of Medicine. Chapel Hill 2017 Approved by: Nathaniel Moorman Mark Heise Marty Ferris Stanley Lemon Bill Marzluff Cary Moody © 2017 Heather Ashley Vincent ALL RIGHTS RESERVED ii ABSTRACT Heather Ashley Vincent: Regulation of mRNA Translation by Human Cytomegalovirus pTRS1 (Under the direction of Nathaniel Moorman) Human cytomegalovirus (HCMV) is a major public health burden. Acute infection during pregnancy can lead to congenital birth defects, and reactivation of a latent infection in immune compromised individuals can cause significant morbidity and mortality. HCMV does not encode its own ribosomes, and is therefore completely reliant on the host translation machinery for viral protein synthesis. HCMV also does not induce host translational shutoff upon infection, thus viral mRNAs must compete with cellular mRNAs to efficiently translate viral proteins. The HCMV protein TRS1 (pTRS1) plays an integral role in translation regulation during HCMV replication by antagonizing the antiviral kinase PKR. Activated PKR phosphorylates eIF2α, which causes an overall inhibition of protein synthesis that inhibits HCMV replication. pTRS1 also increases overall levels of protein synthesis and enhances the translation of reporter mRNAs in a PKR-independent manner, showing that pTRS1 regulates mRNA translation through multiple mechanisms. In this dissertation I sought to define the mechanisms used by pTRS1 to stimulate translation. In chapter 1 I show that pTRS1 inhibits PKR activation by binding to PKR and inhibiting PKR kinase activity. pTRS1 binds PKR residues that are conserved across eIF2α kinases, suggesting that pTRS1 can antagonize multiple eIF2α kinases. In chapter 2 I show that pTRS1 stimulates cap-independent translation. iii pTRS1 enhances the activity of both host and viral internal ribosome entry sites (IRESs) and stimulates translation of a circular mRNA reporter. These pTRS1 functions were independent of its ability to antagonize PKR, but dependent on its ability to bind double- stranded RNA. To understand how pTRS1 stimulates translation, in chapter 4 I identify ribosome-associated, cellular proteins that bind pTRS1. I found that pTRS1 interacts with active protein phosphatase 1 (PP1) catalytic subunits. Rather than affect PP1 catalytic activity, pTRS1 changes the complement of proteins that interact with the PP1 alpha catalytic subunit, possibly to regulate PP1 substrate specificity. Together these data further characterize the mechanisms used by pTRS1 to regulate mRNA translation and reveal how pTRS1 may contribute to the efficient translation of viral mRNAs during HCMV infection. iv To my little man. Your smiling face brightens even the roughest days. v ACKNOWLEDGEMENTS I first want to thank my family for their continued love and support. You have supported me in every step of my journey to becoming a scientist and I am eternally grateful for that. I also want to thank my friends for keeping me sane during my time in graduate school. This was not an easy process, but you were always there for me. I could not have made it without you. I cannot express how grateful I am to have worked with such a wonderful group of people in the Moorman lab. You guys are amazing, and became like family. Thank you for your support and friendship throughout the years. Lastly, none of this would have been possible without the incredible mentorship I received from Nathaniel Moorman. You taught me how to think, and very importantly, to always include proper controls. I am so grateful for the time and effort you put into training me. I am the scientist I am today because of you. Thank you. vi TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ xi LIST OF FIGURES .......................................................................................................... xii LIST OF ABBREVIATIONS AND SYMBOLS ................................................................. xiv CHAPTER 1: INTRODUCTION ........................................................................................ 1 Herpesvirus replication and pathogenesis ............................................................ 1 Human cytomegalovirus pathogenesis ................................................................. 5 Human cytomegalovirus replication ...................................................................... 8 Regulation of mRNA translation during HCMV infection ....................................... 9 Overview of translation regulation ....................................................................... 10 Overview of translation regulation during HCMV infection .................................. 18 The role of the eIF4F complex in HCMV replication ........................................... 19 Alternate modes of translation initiation during infection ..................................... 21 HCMV and eIF2α kinases ................................................................................... 23 Role for HCMV pTRS1 in translation regulation .................................................. 27 Outstanding questions in the field ....................................................................... 31 Figure .................................................................................................................. 35 References .......................................................................................................... 37 CHAPTER 2: MECHANISM OF PKR INHIBITION BY HCMV PTRS1 ........................... 66 Overview ............................................................................................................. 66 vii Introduction ......................................................................................................... 67 Materials and Methods ........................................................................................ 70 Results ................................................................................................................ 77 Discussion ........................................................................................................... 85 Acknowledgements ............................................................................................. 90 Figures ................................................................................................................ 91 Tables.................................................................................................................. 98 References ........................................................................................................ 104 CHAPTER 3: HUMAN CYTOMEGALOVIRUS PTRS1 STIMULATES CAP- INDEPENDENT TRANSLATION ...................................................................... 111 Overview ........................................................................................................... 111 Introduction ....................................................................................................... 112 Materials and Methods ...................................................................................... 115 Results .............................................................................................................. 118 Discussion ......................................................................................................... 123 Acknowledgements ........................................................................................... 125 Figures .............................................................................................................. 126 Tables................................................................................................................ 131 References ........................................................................................................ 132 CHAPTER 4: HCMV PTRS1 INTERACTS WITH ACTIVE PP1 CATALYTIC SUBUNITS ................................................................................... 139 Overview ........................................................................................................... 139 Introduction ....................................................................................................... 140 Materials and Methods ...................................................................................... 142 viii Results .............................................................................................................. 149 Discussion ......................................................................................................... 154 Figures .............................................................................................................. 158 Tables................................................................................................................ 164 References ........................................................................................................ 165 CHAPTER 5: DISCUSSION ......................................................................................... 170 Summary of my findings .................................................................................... 170 Outstanding questions
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