Natural Killer Cell Evasion By Rhesus Cytomegalovirus by Elizabeth Rowland Sturgill A Dissertation Presented to the Department of Molecular Microbiology and Immunology And the Oregon Health and Science University School of Medicine In Fulfillment of the requirements For the degree of Doctor of Philosophy April 2016 TABLE OF CONTENTS ABSTRACT………………………………………………………………………………I ACKNOWLEDGEMENT……………………………………………………………..III PREFACE ……………………………………………………………………………….V SELECTED ABBREVIATIONS…………………………………………………...…VI 1 INTRODUCTION ................................................................................................... 3 1.1 HERPESVIRUSES ................................................................................................. 3 1.1.1 Herpesviridae ................................................................................................... 3 1.1.2 Cytomegalovirus .............................................................................................. 5 1.1.2.1 HCMV Epidemiology and Pathogenesis .................................................... 5 1.1.2.2 CMV Biology .............................................................................................. 6 1.1.2.2.1 Genome Organization ........................................................................... 6 1.1.2.2.2 Virion Structure .................................................................................... 7 1.1.2.2.3 Cellular tropism .................................................................................... 8 1.1.2.2.4 Replication Cycle .................................................................................. 9 1.1.2.2.5 Latency, Persistence & Reactivation .................................................. 11 1.1.2.3 Host Immune Response to CMV Infection ............................................... 12 1.1.2.3.1. Intrinsic Immunity .............................................................................. 13 1.1.2.3.2. Innate Immunity ................................................................................. 13 1.1.2.3.3. Adaptive Immunity ............................................................................ 15 1.1.2.4. CMV Models ............................................................................................ 17 1.1.2.4.1. Rhesus CMV ...................................................................................... 18 1.1.2.4.1.1 Overview ..................................................................................... 18 1.1.2.4.1.2 RhCMV as a model for HCMV .................................................. 19 1.2. NK CELL BIOLOGY .......................................................................................... 20 1.2.1. Overview ....................................................................................................... 20 1.2.2. Lineage, Development & Phenotype ............................................................. 21 1.2.3. NK Cell Receptors ......................................................................................... 23 1.2.3.1. NK Cell Inhibition .................................................................................... 23 1.2.3.2. NK Cell Activation ................................................................................... 27 1.2.4. NK Cell Education ........................................................................................ 33 1.2.4.1. Missing self .............................................................................................. 33 1.2.4.2. Models of NK cell education ................................................................... 34 1.2.5. NK-DC crosstalk ........................................................................................... 38 1.2.6. NK cell memory ............................................................................................. 39 1.3 CMV IMMUNE MODULATION ........................................................................ 41 1.3.1 T cell evasion ................................................................................................. 41 1.3.2 NK cell evasion ................................................................................................. 42 1.3.3. CMV as a vaccine vector .............................................................................. 48 1.4. NK CELLS IN OTHER DISEASE MODELS ..................................................... 49 1.5. HYPOTHESIS ..................................................................................................... 59 2. NATURAL KILLER CELL EVASION IS ESSENTIAL FOR PRIMARY INFECTION BY CYTOMEGALOVIRUS .................................................................. 60 ! 1! 2.1 ABSTRACT ............................................................................................................... 61 2.2 AUTHOR SUMMARY ............................................................................................... 61 2.3 INTRODUCTION ...................................................................................................... 62 2.4 RESULTS ................................................................................................................ 65 2.4.1. RhCMV inhibits cell surface expression of NKG2DLs ................................. 65 2.4.2. MICB is retained in the ER/cis-golgi and associates with Rh159 in RhCMV- infected cells…… ....................................................................................................... 66 2.4.3. Rh159 retains MICB in the ER/cis-golgi ...................................................... 70 2.4.4. HCMV UL148 does not downregulate NKG2DLs ........................................ 74 2.4.5. Deletion of Rh159 restores intracellular maturation of MICB in RhCMV- infected cells……. ...................................................................................................... 76 2.4.6. Deletion of Rh159 increases NK cell stimulation by RhCMV-infected cells 79 2.4.7. Rh159 targets RM NKG2DLS ....................................................................... 81 2.4.8. Rh159 is essential for infection ..................................................................... 82 2.5 DISCUSSION ........................................................................................................... 86 3. RHCMV GENOME ENCODES MULTIPLE GENES RESPONSIBLE FOR NK CELL EVASION ...................................................................................................... 91 3.1. INTRODUCTION ..................................................................................................... 91 3.2. RESULTS ............................................................................................................... 93 3.2.1. RhCMV entry of U373 cells .......................................................................... 93 3.2.2. RhCMV IE2 does not significantly affect NKG2DL expression ................... 95 3.2.3. Rh158-180 encodes additional gene products responsible for downregulation of MICA surface expression ............................................................ 96 3.2.4. Rh191-202 encodes additional gene products responsible for downregulation of MICA surface expression ............................................................ 99 3.3. DISCUSSION ........................................................................................................ 100 4. DISCUSSION AND FUTURE DIRECTIONS ................................................. 103 5. MATERIAL AND METHODS .......................................................................... 114 6. APPENDIX ............................................................................................................. 121 6.1. MS DATA ............................................................................................................. 121 6.2. ALIGNMENT OF RM AND HUMAN NKG2DLS ............................................. 122 6.3. NEXT GEN SEQUENCING DATA .................................................................... 124 ! 2! Chapter 1 INTRODUCTION 2.1 HERPESVIRUSES 1.1.1 Herpesviridae Viruses within the Herpesviridae family are encapsulated, enveloped, double- stranded DNA viruses, with large coding potentials that have co-evolved with their hosts for the past 200 million years. The Herpesviridae family is further divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae, which is based upon duration of replication cycle, cell tropism, sites of latency, and species restriction. A total of eight viruses comprising all three subfamily members of the Herpesviridae family are known to infect humans and can establish persistent infections that cannot be completely cleared by the host. Viruses within the Alphaherpesvirinae subfamily include human pathogens: Herpes simplex virus 1 and 2 (HSV-1, HSV-2) and Varicella-Zoster virus (VZV), which replicate their genomes comparatively quick (hours), infect a wide range of tissues types (mucosal epithelium, neuronal), establish latency in sensory ganglia, and display broad host tropism. The Betaherpesvirinae subfamily members have a slower replication cycle (days), a more restricted host tropism, and include Human Cytomegalovirus (HCMV) and Human herpesvirus 6 and 7 (HHV-6/7). Initial infection can occur in a broad spectrum of tissue types, but can be predominantly ! 3! found in the salivary gland, bone marrow, and spleen, which are also common sites of latency. Members of the Gammaherpesvirinae subfamily, which includes Epstein-Barr virus (EBV) and