Analysis of metabolic alterations in carbon utilization pathways during virus infection Krystal Ann Fontaine A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2014 Reading Committee: Michael Lagunoff, Chair Adam P. Geballe Jason G. Smith Program Authorized to Offer Degree: Microbiology ©Copyright 2014 Krystal Ann Fontaine University of Washington Abstract Analysis of metabolic alterations in carbon utilization pathways during virus infection Krystal Ann Fontaine Chair of the Supervisory Committee: Professor Michael Lagunoff Department of Microbiology Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for their replication. Substantial research has focused on understanding how viruses alter host cellular metabolism in the hopes of identifying metabolic pathways that are critical for successful infection. In this thesis, we explore how two viruses important for biodefense, vaccinia virus (VACV) and dengue virus (DENV), manipulate the global cellular metabolome during infection. In Chapter III, we examine the impact VACV has on the host metabolic network and discover that VACV implements a strikingly unique carbon utilization program during infection. Specifically, we define an important role for glutamine during VACV infection and show that glucose is dispensable for replication. We show that the glutaminolytic pathway of glutamine metabolism is markedly altered in VACV-infected cells and is necessary to replenish the TCA cycle during infection. We further demonstrate that glutaminolysis is required for optimal VACV replication by facilitating the robust viral protein synthesis needed for infectious virion production. In Chapter IV, we find that DENV significantly alters glucose metabolism during infection. In particular, we show that DENV infection activates glycolysis via the upregulation of multiple glycolytic mediators. Moreover, we demonstrate that the glycolytic pathway is essential for efficient DENV replication. Therefore, this body of work reveals how viruses dramatically reprogram central carbon metabolism and offers further evidence that metabolic inhibitors may provide promise in the treatment of virus infections in the future. TABLE OF CONTENTS Page List of Figures ................................................................................................................................ iii List of Tables ................................................................................................................................. iv Chapter I: Introduction .....................................................................................................................1 Viruses and host cellular metabolism ......................................................................................1 Viral-induced changes in central carbon metabolism ..............................................................6 Objectives of this thesis ...........................................................................................................8 Vaccinia virus ..........................................................................................................................9 Dengue virus ..........................................................................................................................12 Hypotheses .............................................................................................................................14 Chapter II: Materials and Methods ................................................................................................17 Cells .......................................................................................................................................17 Inhibitors ................................................................................................................................17 Antibodies ..............................................................................................................................18 Viruses and determination of DENV infection rates .............................................................18 Virus infections for metabolic analysis ..................................................................................19 Cell viability assays ...............................................................................................................20 Nutrient starvation and metabolic inhibitor treatment studies ...............................................20 DENV focus-forming unit reduction assays ..........................................................................22 Glucose uptake assays ............................................................................................................22 Quantitative real-time RT-PCR .............................................................................................23 Western blot analysis .............................................................................................................24 Transmission electron microscopy ........................................................................................25 Statistical analysis ..................................................................................................................26 Chapter III: Vaccinia virus requires glutamine but not glucose for efficient replication ..................................................................................................................27 Summary ................................................................................................................................27 Introduction ............................................................................................................................28 Results ....................................................................................................................................30 Discussion ..............................................................................................................................36 Chapter IV: Dengue virus requires the induction of glycolysis for productive infection ..................................................................................................................52 Summary ................................................................................................................................52 Introduction ............................................................................................................................53 Results ....................................................................................................................................55   i Discussion ..............................................................................................................................60 Chapter V: Conclusions and Future Directions .............................................................................74 Summary ................................................................................................................................74 Conclusions ............................................................................................................................75 VACV ................................................................................................................................75 DENV ................................................................................................................................76 Future Directions ...................................................................................................................77 VACV ................................................................................................................................77 DENV ................................................................................................................................79 References ......................................................................................................................................86                           ii LIST OF FIGURES Figure Number Page 1-1 Viruses alter carbon metabolism in mammalian cells .......................................................16 3-1 Glutamine metabolism is altered during VACV infection ................................................47 3-2 Glutamine is necessary for optimal infectious VACV production ....................................48 3-3 Glutamine is an essential anaplerotic substrate for the TCA cycle during VACV infection ......................................................................................................49 3-4 Glutamine is required to maintain the TCA cycle for VACV protein synthesis ...............................................................................................50 3-5 Glutamine deprivation results in reduced virion formation in VACV-infected cells ......................................................................................................51 4-1 The glycolytic pathway of glucose metabolism is altered during DENV infection ......................................................................................................69 4-2 Glutamine metabolism is perturbed in DENV-infected cells ............................................70 4-3 Exogenous glucose is necessary for optimal infectious DENV production ......................71 4-4 Glycolysis is induced during DENV infection ..................................................................72 4-5 Glycolysis is required for maximal DENV replication ......................................................73 5-1 Glutaminolytic enzyme levels are not elevated during VACV infection ..........................84 5-2 HIF-1α mRNA levels are increased during DENV infection ............................................85   iii LIST OF TABLES Table Number Page 3-1 VACV infection alters global host cellular metabolism ....................................................40 4-1 DENV infection alters global host cellular metabolism ...................................................64   iv ACKNOWLEDGEMENTS There are many people I would like to thank for supporting me during my graduate career. First and foremost, I thank my mentor Michael Lagunoff for his committed guidance in helping me to become an independent scientist. He provided me with clear direction, yet also allowed me the freedom to pursue scientific endeavors that I was excited to explore. I would also like to thank my previous mentors, Sharon Isern and Scott Michael, for the inspiring introduction to research and for the continuous encouragement they have given me over the years. I would simply not be where I am today if it were not for these three individuals. I am also indebted to my committee members (Jason Smith, Adam Geballe, Keith Jerome, and Elaine Raines) for years of advice and insight. Thank you to all the members of the Lagunoff lab, past and present, for helping to make my graduate experience such a memorable one. Specifically, I am beyond thankful for Erica Sanchez and the expansive role she has played in my life. To Roman Camarda, I will continue to try to find the words to convey just how truly grateful I am that you joined the lab. I would also like to express my gratitude to my dear friends, including my fellow classmates, for sharing the path with me on such a monumental journey. Lastly, I am forever grateful for my family and their ferocious love and endless support. To Daddy, Momma, Gramma, and Pop Pop, in particular: thank you, thank you, thank you, thank you.   v DEDICATION I dedicate this dissertation to my Daddy and Momma, the two greatest parents a girl could have hoped for. How lucky I have been and continue to be to have you always by my side. To you both, I love you with all of my heart.   vi