A Study of the Ebola Virus Glycoprotein: Disruption of Host Surface Protein Function and Evasion of Immune Responses
Total Page：16
File Type：pdf, Size：1020Kb
University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2010 A Study of the Ebola Virus Glycoprotein: Disruption of Host Surface Protein Function and Evasion of Immune Responses Joseph R. Francica University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Virology Commons Recommended Citation Francica, Joseph R., "A Study of the Ebola Virus Glycoprotein: Disruption of Host Surface Protein Function and Evasion of Immune Responses" (2010). Publicly Accessible Penn Dissertations. 198. https://repository.upenn.edu/edissertations/198 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/198 For more information, please contact [email protected]. A Study of the Ebola Virus Glycoprotein: Disruption of Host Surface Protein Function and Evasion of Immune Responses Abstract The Ebola virus (EBOV) is a member of the family, Filoviridae, and is the etiological agent of Ebola hemorrhagic fever (EHF). This disease causes significant morbidity and mortality in humans and non- human primates, with human fatality rates reaching 90% during outbreaks of the Zaire subtype. Currently, there are no licensed vaccines or antivirals for EBOV and our understanding of viral pathogenesis is limited. Therefore, further studies examining the pathogenic mechanisms of EBOV are necessary to fully understand and effectively treat EHF. The main Ebola virus glycoprotein (GP) is the only viral protein found on the surface of the Ebola virion and is therefore responsible for mediating attachment and entry of the virus into host cells. However, expression of GP independently of other viral proteins induces dramatic morphological changes including cell rounding and detachment in those cells expressing GP. This phenomenon is referred to as GP-mediated cytopathology and is the focus of the work described herein. We have undertaken studies to identify the mucin domain, a highly glycosylated domain within GP, as sufficiento t cause this cytopathology. We then have used a cell-biological approach to elucidate the mechanism by which this cytopathology occurs. The mucin domain forms a glycan shield at the plasma membrane, disrupting the function of host proteins in the vicinity of GP. We then show that GP-mediated shielding of major histocompatibility complex class I at the cell surface prevents the activation of CD 8+ T cells. Additionally, GP can sterically shield its own epitopes at the cell surface. This model of steric hindrance was also found to apply to the surface of pseudoviral particles, where access to a neutralizing epitope on GP is affected. Our data indicate that the EBOV GP forms a glycan shield with the ability to block antibody binding and disrupt protein function at the cell and virion surface. This study describes a novel viral mechanism for the disruption of surface protein function and suggests a possible mechanism for the evasion of host humoral and cellular immune responses. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Paul Bates, Ph.D. Keywords Ebola virus, glycan shield, MHC I, mucin, adhesion, viral glycoprotein Subject Categories Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/198 A STUDY OF THE EBOLA VIRUS GLYCOPROTEIN: DISRUPTION OF HOST SURFACE PROTEIN FUNCTION AND EVASION OF IMMUNE RESPONSES Joseph Richard Francica A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 Dissertation Supervisor: __________________________ Paul Bates, Ph.D., Associate Professor of Microbiology Graduate Group Chairperson: __________________________ Daniel S. Kessler, Ph.D., Associate Professor of Cell and Developmental Biology Dissertation Committee: Jeffrey M. Bergelson, M.D., Associate Professor of Pediatrics Robert W. Doms, M.D., Ph.D., Professor of Pathology and Laboratory Medicine Michael S. Marks, Ph.D., Associate Professor of Pathology and Laboratory Medicine I would like to dedicate this dissertation to my grandfather, Robert Gauthier. His persistence in his scientific career, the integrity with which he lives his life, and his encouragement of me from a young age has been a guiding influence in my life. ii ACKNOWLEDGMENTS I would like to thank Paul Bates for his mentorship throughout my graduate career. Paul encouraged me to further investigate the initial observations that led to this dissertation, while allowing me the freedom to take my studies in whichever direction interested me. His enthusiasm for scientific discovery and the creativity in his experimental approach has made a deep impression on me as a new scientist. I would like to thank Jeffrey Bergelson, Robert Doms and Michael Marks for their service on my thesis committee and for the genuine interest they have taken in my work. Their advice has helped me focus and streamline my studies, which has undoubtedly condensed the time of my graduate career. I would especially like to acknowledge the mentorship of my program director, Robert Doms, who took personal interest not just in my work, but also in my well being as a graduate student, and who gave me valuable advice in seeking postdoctoral positions. I would like to thank the past and present members of the Bates lab, who have made both direct and indirect contributions to this work. Also, a special thanks to the members of the Doms lab, with whom sharing technical advice, reagents and matters less scientifically related was a daily occurrence. I would like to thank Marisa Bartolomei, Richard Schultz, and Robert Doms for appointing me to the Cell and Molecular Biology and Emerging Infectious Diseases training grants, which financially supported me through my graduate career and allowed me to present my research at several domestic and international meetings. Finally, I would like to thank my family and friends for their constant love and support throughout the highs and lows of my graduate career. iii ABSTRACT A STUDY OF THE EBOLA VIRUS GLYCOPROTEIN: DISRUPTION OF HOST SURFACE PROTEIN FUNCTION AND EVASION OF IMMUNE REPSONSES Joseph Richard Francica Dissertation Supervisor: Paul Bates The Ebola virus (EBOV) is a member of the family, Filoviridae, and is the etiological agent of Ebola hemorrhagic fever (EHF). This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during outbreaks of the Zaire subtype. Currently, there are no licensed vaccines or antivirals for EBOV and our understanding of viral pathogenesis is limited. Therefore, further studies examining the pathogenic mechanisms of EBOV are necessary to fully understand and effectively treat EHF. The main Ebola virus glycoprotein (GP) is the only viral protein found on the surface of the Ebola virion and is therefore responsible for mediating attachment and entry of the virus into host cells. However, expression of GP independently of other viral proteins induces dramatic morphological changes including cell rounding and detachment in those cells expressing GP. This phenomenon is referred to as GP-mediated cytopathology and is the focus of the work described herein. We have undertaken studies to identify the mucin domain, a highly glycosylated domain within GP, as sufficient to cause this cytopathology. We then have used a cell- biological approach to elucidate the mechanism by which this cytopathology occurs. The mucin domain forms a glycan shield at the plasma membrane, disrupting the function of iv host proteins in the vicinity of GP. We then show that GP-mediated shielding of major histocompatibility complex class I at the cell surface prevents the activation of CD 8+ T cells. Additionally, GP can sterically shield its own epitopes at the cell surface. This model of steric hindrance was also found to apply to the surface of pseudoviral particles, where access to a neutralizing epitope on GP is affected. Our data indicate that the EBOV GP forms a glycan shield with the ability to block antibody binding and disrupt protein function at the cell and virion surface. This study describes a novel viral mechanism for the disruption of surface protein function and suggests a possible mechanism for the evasion of host humoral and cellular immune responses. v TABLE OF CONTENTS CHAPTER 1 − EBOLA VIRUS LITERATURE REVIEW AND INTRODUCTION TO EBOLA GLYCOPROTEIN-MEDIATED CYTOPATHOLOGY........................ 1 1.1 Identification of the Ebola virus........................................................................... 1 1.2 Outbreaks and natural reservoirs.......................................................................... 4 1.3 Epidemiology and clinical syndrome................................................................... 7 1.4 Disease pathogenesis............................................................................................ 8 1.5 Treatment and prevention................................................................................... 10 1.6 Genomic organization, viral proteins and virion structure................................. 11 1.7 Glycoprotein processing, function and structure................................................ 14 1.8 Viral entry........................................................................................................... 18 1.9 Genome replication