Ebola Virus RNA Editing: Characterization of the Mechanism and Gene Products. by Masfique Mehedi B.Sc., M.Sc. A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Medical Microbiology University of Manitoba Winnipeg Copyright © 2012 by Masfique Mehedi COPYRIGHT PERMISSION A thesis/practicum submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfilment of the requirement of the degree of DOCTOR OF PHILOSOPHY © Masfique Mehedi, September 2012 Permission has been granted to the library of the University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies to the film, and to University Microfilms Inc. to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. ii THIS THESIS HAS BEEN EXAMINED AND APPROVED. Thesis supervisor, Dr. Heinz Feldmann, M.D., Ph.D. Associate Professor of Medical Microbiology, University of Manitoba & Chief, Laboratory of Virology, Rocky Mountain Laboratories, DIR, NIAID, NIH, Hamilton, MT Dr. Kevin Coombs, Ph.D. Professor of Medical Microbiology & Associate Dean (Research), Faculty of Medicine, University of Manitoba Dr. Steven Pind, Ph.D. Assistant Professor, Biochemistry and Medical Genetics, University of Manitoba External Examiner, Dr. Paul A Rota, Ph.D. Adjunct Professor, Emory University & University of Georgia, & Measles Team Lead, MMR and Herpes virus Laboratory Branch, Centers for Disease Control and Prevention (CDC), Atlanta, GA iii ABSTRACT EBOLA VIRUS RNA EDITING: CHARACTERIZATION OF THE MECHANISM AND GENE PRODUCTS. Masfique Mehedi, B.Sc., M.Sc. University of Manitoba, 2012 Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. iv Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene- specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention. v ACKNOWLEDGEMENTS This has been a wonderful learning endeavor with the Department of Medical Microbiology, University of Manitoba. I would like to thank my dear committee members Kevin Coombs and Steven Pind for their comments and guidance over the years. I would like to extend my sincere gratitude toward my external reviewer, Paul A Rota (CDC, Atlanta, GA) for reviewing my thesis. I have been extremely fortunate to have Heinz Feldmann as my supervisor. It is difficult to express in words, but definitely I owe a huge debt of gratitude to him for his patient guidance, precious encouragement, valuable advice, and tremendous support in my graduate study and helping me bloom my research potential. I would like to thank members of the Special Pathogens Program, National Microbiology Lab, Winnipeg for providing generous support; especially, Darryl Falzarano for his unconditional support in my research training, Thomas Hoenen, Allison Groseth, and Marko Zivcec for their cloning lessons that helped me in my research, and Hideki Ebihara for providing minigenome plasmids and his valuable suggestion in my research. I am also thankful to Jochen Seebach & Hans-Joachim Schnittler, University of Munster, Germany for their help with the experimental procedures of the transendothelial vi resistance (TER) measurement and to Garett Westmacott & Keding Cheng for their help with the mass spectrometry. I am grateful to the National Institutes of Health for the Pre-Doctoral Fellowship to do research at the Rocky Mountain Laboratories (RML), Hamilton, MT. My heartiest thanks go toward the members of the Laboratory of Virology for their numerous supports in my research, especially Ricki Feldmann for the biosafety level 4 training and Shelly Robertson for helping with the flow cytometer. I am thankful to Stacy Ricklefs, Research Technology Branch, RML for help in the development of the rapid transcript quantification assay. I am obliged to the University of Manitoba for providing me the graduate student entrance scholarships (2006-07), graduate student scholarships (2008), and Dean’s award (2008) in the Manitoba Health Research Poster Competition; to the Canadian Institutes of Health Research (CIHR) for the Silver award (2008) in the CIHR poster competition; to the American Society of Virology (ASV) for the travel award (2011) to attend the ASV conference. Last, but not least, I am grateful to my wife (Farhana Yesmen) for being patient and providing constant encouragements. vii DEDICATION This thesis is dedicated to my beloved parent. viii TABLE OF CONTENTS Page Copyright Permission ii Approval iii Abstract iv Acknowledgements vi Dedication viii Table of Contents ix List of Tables xiv List of Figures xiv List of Appendices xvi List of Copyrighted Materials xvii List of Abbreviations xviii 1. INTRODUCTION 1 1.1 History of filoviral hemorrhagic fever 1 1.2 Filovirus classification 7 1.2.1 Virus taxonomy 7 1.2.2 Biohazard classification 7 1.3 Filovirus ecology, natural reservoir, and transmission 9 1.4 Ebola hemorrhagic fever (EHF) 11 1.4.1 Disease symptoms, pathology, and diagnosis 11 1.4.2 Treatment and prevention 14 ix 1.4.3 Vaccine development 16 1.5 EBOV morphology and structure 18 1.6 EBOV genome 19 1.7 EBOV life cycle 23 1.7.1 Attachment and entry 23 1.7.2 Transcription and replication 28 1.7.3 Budding 29 1.8 EBOV pathogenesis and evasion of host immune response 29 1.9 EBOV proteins 31 1.9.1 Nucleoprotein (NP) 31 1.9.2 Viral protein (VP) 35 32 1.9.3 Viral protein (VP) 40 33 1.9.4 Viral protein (VP) 24 34 1.9.5 Viral protein (VP) 30 35 1.9.6 RNA dependent RNA polymerase (L) 36 1.9.7. Glycoprotein (GP) 37 1.9.7.1 Soluble glycoprotein (sGP) 37 1.9.7.2 Δ-peptide 38 1.9.7.3 Transmembrane glycoprotein (GP1,2) 40 1.10 RNA editing 41 1.10.1 EBOV RNA editing 43 1.11 Objective and Hypothesis 45 1.11.1 Significance 45 1.11.2 Hypotheses 46 1.11.3 Objectives 46 1.11.4 Justification 47 2. MATERIALS AND METHODS 48 x 2.1 Cells and media 48 2.1.1 Maintenance of cell lines 48 2.1.2 Isolation and culture of primary cells 48 2.1.2.1 Macrophages 48 2.1.2.2 Neutrophils 49 2.1.2.3 HUVECs 50 2.1.3 Bacterial cells 51 2.2 Virus infection 51 2.2.1 Virus strains 51 2.2.2 In vitro infection 51 2.3 Cloning and site-directed mutagenesis 52 2.3.1 r.ssGP and r.sGP expression plasmids 52 2.3.2 Cys53 mutated r.ssGP 53 2.33. Minigenome 54 2.3.4 Dual-reporter minigenome 56 2.3.5 Mutations and deletions 58 2.4 r.ssGP and r.sGP production 61 2.5 ssGP transcript detection 62 2.6 Protein analysis 63 2.6.1 Removal of carbohydrates 64 2.6.2 SDS-PAGE 65 2.6.3 2-D gel electrophoresis 65 2.6.4 Protein detection 66 2.6.5 2-D-DIGE 66 2.6.6 Mass spectrometry 67 2.7 ssGP function 69 2.7.1 Neutrophil binding 69 xi 2.7.2 Barrier function analysis of endothelial cells 69 2.7.2.1 Chamber slides preparation 69 2.7.2.2 Impedance spectroscopy 70 2.8 Detection of RNA editing 73 2.8.1 Development of RTQA 73 2.9 Characterization of RNA editing 77 2.9.1 Minigenome rescue 77 2.9.2 In vitro-transcription of minigenome DNA 77 2.9.3 Detection and quantification of reporter proteins 78 2.9.3.1 Confocal microscopy 78 2.9.3.2 Epi-fluorescence microscopy 78 2.9.3.3 FACS 80 2.10 Statistical analysis 80 2.11 Ethics and biosafety statements 80 3.