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The Dissertation Committee for Felicia Gilfoy Santa Maria Guerra Certifies that this is the approved version of the following dissertation: West Nile virus versus the host cell: Identification of factors that modulate infection Committee: Peter Mason, PhD, Supervisor Nigel Bourne, PhD Robert Davey, PhD Don M Estes, PhD Gregg Milligan, PhD Frank Scholle, PhD __________________ Dean, Graduate School West Nile virus versus the host cell: Identification of host factors that modulate infection by Felicia Gilfoy Santa Maria Guerra, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas Medical Branch December 2008 Dedication To my family and friends who have helped me and encouraged me throughout my graduate career and life. A special ‗shout-out‘ goes to Bridget, who has been a constant friend since we first started school. You helped me open my shell and I will never forget your part in me meeting Sergio! To my husband and friend, Sergio, who has been there for me through all the highs and lows and continues to be my source of strength and motivation. Acknowledgements This work could not have been completed without the help of numerous individuals. The collaborative and intellectually-stimulating environment created by the faculty, post-docs and students at the University of Texas Medical Branch has allowed me to grow as a scientist and as a person. I am thankful for the guidance and support of my committee. Their patience and willingness to help was instrumental in my success as a graduate student. I thank everyone who donated time and/or reagents for this dissertation—a lot of this could not have been done without it. I send a big thank you to past and present members of the Mason lab. They have all made my experience here at UTMB a unique and pleasant one. I am very grateful to Rafik Fayzulin, who toiled along-side me during those long and monotonous siRNA screens and would particularly like to thank Shannan and Doug, two fellow students in the lab, who helped make the passing time a little more enjoyable. It is always nice when you have comrades with which to share the highs and lows of the graduate experience. Last but not least, I thank my mentor, Peter, for your patience and guidance. I have learned so much under your tutelage and have grown immensely as a young scientist. I am fully aware of the time and effort it takes to mentor a graduate student and I am grateful you were willing to invest it in me. This dissertation was made possible, in part, through grants provided to Dr Peter W. Mason and my funding through National Institutes of Health Training Grant in Emerging and Tropical Infectious Diseases. iv West Nile virus versus the host cell: Identification of host factors that modulate infection Publication No._____________ Felicia Gilfoy Santa Maria Guerra, PhD The University of Texas Medical Branch, 2008 Supervisor: Peter W Mason There are two competing aspects of virus-host interactions: (i) those that enhance virus transmission and (ii) those that reduce or prevent viral replication and transmission. Each of these types of interactions is critical and both must be investigated to fully understand viral pathogenesis. Cells encode a variety of molecules which are critical for recognizing viral infections. One such molecule, the dsRNA sensor PKR, has been shown to be important for IFN- induction. Therefore, to determine whether PKR was involved in the recognition of WNV infection, cells lacking PKR were infected with WNV and assayed for IFN production. Interestingly, PKR-null cells demonstrated dramatically lower levels of WNV-induced IFN compared to wild type cells. Additionally, chemical inhibition of PKR activity or post-translational gene silencing of PKR expression severely impaired WNV-induced IFN production, suggesting that PKR is critical for the induction of IFN following WNV infection. Further analysis suggested that PKR may be important for the activation of NFB, suggesting a possible mechanism of IFN- v induction. Consistent with cell line data, PKR was shown to be critical for WNV-induced IFN production in primary mouse bone marrow-derived dendritic cells. The recognition of WNV is an important aspect of controlling infection; however, it is only one side of the story. Host factors which WNV utilizes to facilitate its infection and replication are also key to understanding viral pathogenesis. The presence or absence of specific factors may control the level of viral replication, host tropisms and, ultimately, viral pathogenesis. To identify host co-factors that are essential for WNV infection and/or replication, small interfering RNAs (siRNAs) were used to systematically knockdown host gene products and levels of WNV infection and/or replication was assayed in the absence of these factors. A siRNA library screen identified ten cellular proteins which are essential for WNV infection and/or replication. Two of these genes encoded subunits of the proteasome. Chemical inhibitors of proteasome activity confirmed that the proteasome is critical for efficient WNV replication. vi Table of Contents List of Tables ....................................................................................................... xiii List of Figures ...................................................................................................... xiv CHAPTER 1: INTRODUCTION 1 Family Flaviviridae ..................................................................................................1 Genus Hepacivirus ..........................................................................................2 Genus Pestivirus .............................................................................................3 Genus Flavivirus .............................................................................................4 Genome organization .............................................................................5 Flavivirus proteins .................................................................................8 Flavivirus lifecycle...............................................................................18 West Nile Virus......................................................................................................23 History of WNV and recent outbreaks..........................................................23 Taxonomy .....................................................................................................27 WNV transmission ........................................................................................29 Mosquito transmission .........................................................................30 Non-vectored transmission ..................................................................32 WNV disease and pathology .........................................................................34 Avians ..................................................................................................35 Rodent models .....................................................................................37 Non-human primates ............................................................................39 Humans ................................................................................................40 The Innate Immune Response: Type I Interferon ..................................................41 Induction of type I interferon ........................................................................42 Key players ..........................................................................................42 Signaling pathways ..............................................................................50 Type I interferon signal transduction ............................................................54 vii Impetus for This Research .....................................................................................55 Hypothesis and goals ....................................................................................56 CHAPTER 2: THE DOUBLE-STRANDED RNA-DEPENDENT PROTEIN KINASE, PKR, IS A CRITICAL FACTOR FOR WEST NILE VIRUS INCUDED IFN SYNTHESIS IN VITRO 58 Abstract ..................................................................................................................58 Introduction ............................................................................................................59 Flaviviruses and the innate immune response ..............................................59 PKR as a factor of the innate immune response ...........................................62 Replicons and virus-like particles .................................................................67 Replicons..............................................................................................67 Virus-like particles ...............................................................................68 Rationale .......................................................................................................70 Materials and Methods ...........................................................................................71 Cell Lines used and their maintenance .........................................................71 WNV virus like particle (VLP) preparation..................................................72 Virus and VLPs utilized ................................................................................72 Virus