Latent and Lytic KSHV Infection Require Altered Host Cell Metabolism Erica Lee Sanchez A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2016 Reading Committee: Michael Lagunoff, Chair Adam P. Geballe Nina R. Salama Program Authorized to Offer Degree: Molecular and Cellular Biology © Copyright 2016 Erica Lee Sanchez University of Washington Abstract Latent and Lytic KSHV Infection Require Altered Host Cell Metabolism Erica Lee Sanchez Chair of the Supervisory Committee: Professor Michael Lagunoff Microbiology Viruses are obligate intracellular parasites that depend on host cell machinery for their production and spread. Host metabolism is dramatically altered by several viruses to provide metabolic intermediates for biosynthetic as well as bioenergetic precursors. Understanding how a virus establishes and depends on a specific metabolic signature is critical to revealing possible therapeutic targets of infected cells. In this thesis, I further examined metabolic alterations during infection with the oncogenic virus, Kaposi’s Sarcoma Associated Herpesvirus (KSHV). Like all herpesviruses, KSHV has both a latent and lytic viral life stage. Both latent and lytic cells are found in the KS tumor, therefore, I identified the metabolic alterations in both latent and lytic cells. Previous research indicated that glycolysis and fatty acid synthesis (FAS) were required during latent KSHV infection for the survival of latently infected endothelial cells. In Chapter 1, I confirmed the requirement for FAS during latent infection. After presenting the background (Chapter 1) and Methods (Chapter 2), in Chapter 3, I defined the critical role glutamine metabolism plays in endothelial cells infected with KSHV. I also identify the cellular mechanism by which glutamine addiction is established during latency. Finally, in Chapter 4, I demonstrated that glycolysis, glutamine metabolism, and FAS are all required for maximal infectious KSHV virion production in lytically replicating cells. We show that each of these metabolic pathways contribute to virus production at a different step in lytic replication. Therefore, this body of work provides evidence that both latent and lytic KSHV infection depend on central carbon metabolism. These data may support the development or improvement of clinical therapies that target these carbon utilization pathways, thereby treating all infected cells of the KS tumor. TABLE OF CONTENTS List of Figures ............................................................................................................................................................................. iv List of Tables ................................................................................................................................................................................ v Chapter 1. Introduction ........................................................................................................................................................... 1 1.1 Herpesviruses ........................................................................................................................................................ 1 1.2 KSHV and KS ........................................................................................................................................................... 2 1.3 KSHV Latent and Lytic Cell Culture Systems ............................................................................................ 6 1.4 Altered Metabolism and Viral Induced Oncogenesis ............................................................................ 8 1.4.1 Cancer Cell Metabolism ................................................................................................................................ 8 1.4.2 Viruses and Host Cell Metabolism ........................................................................................................... 9 1.4.3 KSHV and Host Cell Metabolism ............................................................................................................ 10 1.5 Hypothesis ............................................................................................................................................................ 13 Chapter 2. Materials and Methods .................................................................................................................................. 18 2.1 Cell Culture Systems ......................................................................................................................................... 18 2.2 Viruses .................................................................................................................................................................... 18 2.3 Latent KSHV Infections ................................................................................................................................... 19 2.4 Immunofluorescence ....................................................................................................................................... 20 2.5 Reagents ................................................................................................................................................................ 20 2.6 Glutamine Uptake Assay ................................................................................................................................. 21 2.7 Glutamine Starvation, BPTES, and GPNA Treatment Studies ........................................................ 21 2.8 Western Blot Analysis ..................................................................................................................................... 22 2.9 Quantitative RT-PCR ........................................................................................................................................ 23 2.10 siRNA Transfection and Cell Survival ....................................................................................................... 23 2.11 Lytic KSHV Assay ............................................................................................................................................... 24 2.12 Statistical analysis ............................................................................................................................................. 24 Chapter 3. Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival ................................................................................................................................................ 25 3.1 Abstract .................................................................................................................................................................. 25 i 3.2 Authors Summary ............................................................................................................................................. 26 3.3 Introduction ......................................................................................................................................................... 27 3.4 Results .................................................................................................................................................................... 29 3.4.1 Latent KSHV Infection of Endothelial Cells Induces Increased Glutamine Uptake ......... 29 3.4.2 Exogenous Glutamine is Required for the Survival of Endothelial Cells Latently Infected with KSHV ........................................................................................................................................................................ 30 3.4.3 Glutamine Starvation Leads to Apoptosis of KSHV Infected Endothelial Cells ................. 31 3.4.4 Glutaminolysis is Required for KSHV Infected Endothelial Cell Survival ............................ 32 3.4.5 KSHV Induces Expression of the Myc/MondoA Network and Their Targets, Including the Glutamine Transporter SLC1A5 ..................................................................................................................... 33 3.4.6 The Glutamine Transporter SLC1A5 is Required for Survival of Endothelial Cells Latently Infected with KSHV ................................................................................................................................... 34 3.4.7 MondoA Regulation of Glutaminolysis is Required for Survival of Endothelial Cells Latently Infected with KSHV ................................................................................................................................... 35 3.5 Discussion ............................................................................................................................................................. 36 Chapter 4. Host Metabolism Is Required for Maximal KSHV Virion Production ......................................... 48 4.1 Abstract .................................................................................................................................................................. 48 4.2 Introduction ......................................................................................................................................................... 49 4.3 Results .................................................................................................................................................................... 51 4.3.1 Glycolysis is Required for Maximal KSHV Virion Production ................................................... 51 4.3.2 Glutaminolysis is Required for Maximal KSHV Virion Production ......................................... 53 4.3.3 Fatty Acid Synthesis is Required for Maximal KSHV Virion Production .............................. 54 4.3.4 Glycolysis is Required for Early and Late Gene Expression ...................................................... 54 4.3.5 Glutamine Metabolism is Required for Late KSHV Gene Expression .................................... 55 4.3.6 Fatty Acid Synthesis is Not Required for Early or Late KSHV Gene Expression ............... 55 4.3.7 Glycolysis and Glutamine Metabolism, but Not Fatty Acid Synthesis, are Required for Early and Late Protein Synthesis .......................................................................................................................... 56 4.4 Discussion ............................................................................................................................................................. 57 Chapter 5. Conclusions and Future Directions .......................................................................................................... 67 5.1 Summary ............................................................................................................................................................... 67 5.2 Latent KSHV Infection Requires Glutamine Metabolism Via Glutaminolysis .......................... 68 ii 5.3 Carbon Metabolism and Lytic KSHV Infection ...................................................................................... 69 5.4 Future Directions ............................................................................................................................................... 70 5.4.1 Latent KSHV and Glutaminolysis ........................................................................................................... 70 5.4.2 Lytic KSHV and Cellular Metabolism ................................................................................................... 72 5.5 Conclusions .......................................................................................................................................................... 74 Copyright Permissions ......................................................................................................................................................... 78 Bibliography .............................................................................................................................................................................. 82 iii LIST OF FIGURES Figure 1.1: Virus infection alters host cell metabolism 14 Figure 1.2: KSHV-infected cells induce the formation of lipid droplet organelles 16 Figure 1.3: Palmitate rescues TOFA-induced cell death in KSHV latently infected endothelial cells 17 Figure 3.1: Glutamine uptake is increased by latent KSHV infection 40 Figure 3.2: Glutamine metabolism is required for survival of latently infected endothelial cells 41 Figure 3.3: Glutamine starvation leads to apoptosis of KSHV-infected endothelial cells 42 Figure 3.4: Glutamine is required for glutaminolysis in KSHV-infected endothelial cells 43 Figure 3.5: KSHV infection of endothelial cells increases protein expression of the Myc/MondoA network and downstream targets, including the glutamine transporter SLC1A5 44 Figure 3.6: Endothelial cells latently infected with KSHV require the glutamine transporter SLC1A5 for survival 45 Figure 3.7: MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV 46 Figure 3.8: Schematic of glutamine metabolism via glutaminolysis during KSHV infection of endothelial cells 47 Figure 4.1: Glycolysis is required for maximal KSHV virion production 59 Figure 4.2: Glutaminolysis is required for maximal KSHV virion production 60 Figure 4.3: Fatty Acid Synthesis is required for maximal KSHV virion production 61 Figure 4.4: Glycolysis is required for early and late KSHV gene expression 62 Figure 4.5: Glutamine metabolism is required for late KSHV gene expression 63 Figure 4.6: Fatty Acid Synthesis is not required for early or late KSHV gene expression 64 Figure 4.7: Glycolysis and Glutamine metabolism are required for KSHV protein synthesis 65 Figure 4.8: Carbon metabolism and KSHV virus production 66 Figure 5.1: Metabolic targets of both latent and lytic KSHV infection exist 75 Figure 5.2: Solute carrier transport proteins, SLC3A2 and SLC7A5, are upregulated by KSHV infection 76 Figure 5.3: Virion assembly appears unchanged by FAS inhibition 77 iv LIST OF TABLES Table 1.1: Virus Induced Metabolism 15 v ACKNOWLEDGEMENTS I would like to thank several people for their support throughout my graduate school career. To my PI and mentor, Michael Lagunoff, for being patient with me as I worked to become an independent scientist. Thank you for allowing me to make the most of my graduate school experience both at the bench and outside of lab. Your guidance has truly helped me identify and define my scientific and professional career goals. To my labmates, past and present, thank you for making lab supportive and fun, and for sharing your scientific ideas and feedback. I would specifically like to thank my previous lab mates and in-lab mentors, Tracie Delgado and Krystal Fontaine. Thank you Tracie for showing me the ropes early on, helping me fall in love with metabolism, and for introducing me to the SACNAS community. To Krystal, thank you for always dancing with me in celebration of the good times and to work through some of those tough times. You ladies have both been by my side as mentors, scientific role models, and friends. I will value our relationships always. To my students, especially miss Hanna Hong and miss Angel Thalhofer. Thank you for being the most dedicated mentees a grad student mentor could ask for and for color-coding your data for me. So proud of both of you! To my MCB and Micro classmates, thank you for your support at each and every graduate school milestone. I am very lucky to have such strong colleagues that I also call friends! To the MCB program, especially MaryEllin and our program directors, for almost always saying ‘Yes’ to requests to improve and support my UW experience and the experience of all STEM students. Thank you for making this experience special and memorable! To the UW SACNAS and GO-MAP communities, thank you for providing a space for me to grow academically and professionally, while also helping me establish an important work-life balance. To my friends from Davis, especially the ladies of 921 Gregory Place. I love you all. BFL! Finally, and most importantly, thank you to my family. Especially Mom, Dad, Ava, Grandma, Uncle Billy and Aunt Betz. Without you, this would not have been possible. Thank you for believing in me, for cheering me on and for joining me in celebrating this accomplishment. I love you all! vi