UNIVERSITY OF CALIFORNIA RIVERSIDE A Novel Role for SCAMP3 as an Innate Immune Factor A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Microbiology by Vladimir Radic March 2017 Dissertation Committee: Dr. I-Chueh Huang, Co-Chairperson Dr. Rong Hai, Co-Chairperson Dr. Ilhem Messaoudi Copyright by Vladimir Radic 2017 The Dissertation of Vladimir Radic is approved: Committee Co-Chairperson Committee Co-Chairperson University of California, Riverside ABSTRACT OF THE DISSERTATION A Novel Function of SCAMP3 as an Innate Immune Factor by Vladimir Radic Doctor of Philosophy, Graduate Program in Microbiology University of California, Riverside, March 2017 Dr. I-Chueh Huang, Co-Chairperson Dr. Rong Hai, Co-Chairperson Influenza A Viruses (IAV’s) pose a significant threat to public health. On average, Influenza infections account for 25,000 deaths and 250,000 hospitalizations every year in the United States. Despite having been discovered in the 1930’s, and despite decades of study influenza viruses continue to cause significant morbidity and mortality. Furthermore, with the emergence of Highly Pathogenic Avian Influenza Viruses (HPAIV) in humans, IAV’s pose a greater threat than ever before. The innate immune response is critical to controlling these viral infections, but despite over half a century passing since the discovery of the first innate immune proteins, the full scope of innate immunity has yet to be elucidated. The Interferon-Induced Transmembrane (IFITM) family of proteins has been shown to strongly restrict a broad range of viruses, iv including IAV5,6,7. However, the exact mechanism of this inhibition as well as the mechanism of IFITM regulation and metabolism is poorly understood. Secretory Membrane Protein 3(SCAMP3) was first identified in 1997 and has since been implicated in EGFR recycling and Salmonella pathogenesis. Here we demonstrate a new function of SCAMP3 in the innate immune response. We show that SCAMP3 can function to stabilize IFITM3 by decreasing its lysosomal degradation. We show that SCAMP3 can alter the trafficking of IFITM, resulting in the protein being recycled from early endosomes back to the Golgi apparatus. Upon SCAMP3 depletion, this recycling is ablated and IFITM3 undergoes increased lysosomal degradation. We also show that SCAMP3 can directly restrict Influenza A Viruses. We show that SCAMP3 and interfere with cleavage of Highly Pathogenic Avian Influenza (HPAIV) Hemagglutinin (HA) cleavage, resulting in viruses that are deficient in their ability to enter target cells. We also show that SCAMP3 expression, phosphorylation and localization is altered upon viral infection or stimulation with various innate immune proteins, suggesting that SCAMP3 is an innate immune effector. This research deepens our understanding of the innate immune response and presents a potential therapeutic strategy for treatment or prophylaxis of viral disease. v Table of Contents Chapter 1: Introduction .......................................................................................................... 1 1.1 Influenza Viruses ......................................................................................................................... 2 1.2 History of Influenza ................................................................................................................... 3 1.3 Influenza Structure and Classification................................................................................. 8 1.4 The Influenza Genome ............................................................................................................. 10 1.5 The Influenza Life Cycle .......................................................................................................... 12 1.6 The Hemagglutinin Protein ................................................................................................... 18 1.7 Innate Immunity to Influenza Viruses ............................................................................... 22 1.8 Abrogation of Innate Immunity by Influenza Viruses .................................................. 24 1.9 Adaptive Immunity to Influenza Viruses .......................................................................... 26 1.10 Antiviral Drugs ........................................................................................................................ 28 1.11 The Interferon Induced Transmembrane Proteins ................................................... 31 1.12 The Discovery of IFITM Antiviral Activity ...................................................................... 32 1.13 IFITM Protein Structure ....................................................................................................... 34 1.14 Post Translational Modifications of IFITM Proteins .................................................. 66 1.15 Models of IFITM Mediated Viral Restriction ................................................................. 37 1.16 SCAMP3 ....................................................................................................................................... 39 1.17 References ................................................................................................................................. 45 Chapter 2: SCAMP3 Regulates IFITM3 Expression ..................................................... 58 2.1 Introduction ................................................................................................................................ 62 2.2 Results ........................................................................................................................................... 58 2.3 Discussion .................................................................................................................................... 62 2.4 Materials and Methods ............................................................................................................ 68 2.5 Figures ........................................................................................................................................... 76 2.6 References ................................................................................................................................... 90 Chapter 3: SCAMP3 Is and Innate Immune Factor which Inhibits IAV infection by altering Hemagglutinin Trafficking. .......................................................................... 94 3.1 Introduction ................................................................................................................................ 95 3.2 Results ........................................................................................................................................ 100 3.3 Discussion ................................................................................................................................. 106 3.4 Materials and Methods ......................................................................................................... 109 3.5Figures ......................................................................................................................................... 116 References ........................................................................................................................................ 121 Chapter 4: Discussion ....................................................................................................123 vi List Figures Chapter 1 Figure 1.1 H5N1 Death Rates.......................................................................................................... 2 Figure 1.2 Spanish Flu Death Rates ............................................................................................ 3 Figure 1.3 Influenza Structure ....................................................................................................... 9 Figure 1.4 The Influenza Life Cycle ............................................................................................ 12 Figure 1.5 Hemagglutinin Mediated Fusion ............................................................................ 19 Figure 1.6 Hemagglutinin Sequence Comparisons ............................................................... 21 Figure 1.7 Innate Immunity to Influenza Viruses ................................................................. 23 Figure 1.8 The IFITM Proteins ..................................................................................................... 31 Figure 1.9 IFITM Topology ............................................................................................................ 35 Figure 1.10 IFITM Restriction Mechanism .............................................................................. 38 Figure 1.11 SCAMP3 ......................................................................................................................... 40 Figure 1.12 SCAMP3 Relocalizes during Salmonella Infection ........................................ 32 Chapter 2 Figure 2.1 SCAMP3 Interacts with IFITM3 ............................................................................... 76 Figure 2.2 SCAMP3 Contributes to IFITM Restriction of Viral Entry ............................ 78 Figure 2.3 SCAMP3 Stabilizes IFITM3 ........................................................................................ 80 Figure 2.4 SCAMP3 Contributes to IFITM Localization to the Golgi Network ............. 82 Figure 2.5 SCAMP3 Regulates Early-endosome