Structure-Function Analysis of Ebola Virus Glycoproteins by Darryl L. Falzarano B.Sc., M.Sc. A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Medical Microbiology University of Manitoba Winnipeg, MB Copyright © 2010 by Darryl L. Falzarano THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES ***** COPYRIGHT PERMISSION A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree of Doctor of Philosophy (c) 2010 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 of 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. i THIS THESIS HAS BEEN EXAMINED AND APPROVED. ______________________________________________________ Thesis supervisor, Dr. Heinz Feldmann, M.D., Ph.D. Associate Professor of Medical Microbiology Chief, Laboratory of Virology, Rocky Mountain Laboratories, DIR, NIAID, NIH ______________________________________________________ Dr. Kevin Coombs, Ph.D. Professor of Medical Microbiology, Internal Medicine Associate Dean (Research) Faculty of Medicine ______________________________________________________ Dr. John Wilkins, Ph.D. Professor of Internal Medicine, Immunology, Biochemistry and Medical Genetics Director, Manitoba Centre for Proteomics and Systems Biology ______________________________________________________ External Examiner, Dr. Anthony Sanchez, Ph.D. Health Scientist Centers for Disease Control and Prevention, Atlanta, GA ______________________________ Date ii ACKNOWLEDGEMENTS I am grateful to the Department of Medical Microbiology and all its members for their support over the course of my graduate degrees. I would like to thank my committee members, Kevin Coombs and John Wilkins for their contributions, guidance and criticism. Hopefully it made a difference. I would also like to thank my external reviewer, Anthony Sanchez (CDC, Atlanta, GA) for accepting to review my thesis. To all the member members of Special Pathogens program, especially the many members of the BIG lab, thanks for your inspiration, assistance and attempts to improve my “friendliness factor” (however misguided you may be). I would like to thank my supervisor, Heinz Feldmann, for providing me some exceptional opportunities to learn more about science, myself and what is right. I appreciate your open-door policy (I think I put it to good use), your candid manner (if an abstract is bad… it’s bad… what else is there to say?) and how you treat everyone with a great deal of respect. Thanks to Ricki for BSL4 training… and all the TCID50 fun. I am indebted to Oleg Krokhin (Manitoba Centre for Proteomics and Systems Biology) for his expert mass spectrometry skills and enthusiasm to further investigate post-translational modifications that others would not even notice. I would also like to thank Ayato Takada (Hokkaido University, Sapporo, Japan) and Xiangao Qiu (Public Health Agency of Canada, Winnipeg, MB) for supplying the anti-ZEBOV monoclonal antibodies Z42/3.7 and P1302H11, respectively. I would especially like to thank: Hideki Ebihara, for sharing some of his seemingly endless knowledge of all things viral; Hans Schnittler and Jochen Seebach for the opportunity to study endothelial cells in Dresden (it was quite an experience – and yes iii Canadians can handle German beer); and Ute Ströher, Allison Groseth and Thomas Hoenen for their input into these projects over the years. I would like to thank the MHRC for graduate studentships in 2007/08 and 2008/09, in addition to the Faculty of Graduate Studies, Department of Medical Microbiology, American Society for Tropical Medicine and Hygiene and the International Congress of Virology for travel scholarships that helped support conference attendance. To my wife, Kirsten, without whom I never would have done this (yeah… thanks a lot), for putting up with me while this was being written, our house was being renovated and right… we also had a son. To Noah, for sleeping through the night at 2 months and being good for mom – most of the time. To my parents, for everything… THANK-YOU. iv TABLE OF CONTENTS Page Copyright Permission…………………………………………………………… i Approval………………………………………………………………………… ii Acknowledgements……………………………………………………………... iii Table of Contents……………………………………………………………….. v List of Tables…………………………………………………………………… viii List of Figures…………………………………………………………………... ix List of Copyrighted Materials…………………………………………………... xi List of Abbreviations…………………………………………………………… xii Abstract………………………………………………………………………… xvi 1. INTRODUCTION Page 1.1 Virus Taxonomy and Particle Structure 1 1.2 Transmission and Clinical Symptoms 4 1.3 Outbreaks of Ebola Virus 7 1.4 Ebola Virus Life Cycle 10 1.4.1 Attachment 10 1.4.2 Entry 15 1.4.3 Genome replication 19 1.4.4 Virus assembly and release 21 1.5 Biosynthesis and Structure of the Glycoprotein Products 22 1.6 Pathogenesis of Ebola virus infection 30 1.6.1 Role of the endothelium in Ebola hemorrhagic fever 37 1.6.2 Disseminated intravascular coagulation 40 1.7 Functions of the Soluble Glycoproteins 43 1.7.1 Direct functions of the soluble glycoproteins 43 1.7.2 sGP indirectly limits GP1,2 expression and cytotoxicity 46 1.8 Role of Glycosylation in Protein Structure and Function 47 1.9 Objectives and Hypothesis 53 1.9.1 Significance 53 1.9.2 Hypothesis 54 1.9.3 Objectives 54 2. MATERIALS AND METHODS 2.1 Isolation and Maintenance of Cells 56 2.1.1 Maintenance of cell lines 56 2.1.2 Isolation and culture of human umbilical vein endothelial cells 56 2.2 Virus Infection 57 2.2.1 Virus strains 57 2.2.2 Determination of virus titre by immunoplaque 58 2.3 Cloning and Site-Directed Mutagenesis 59 v 2.3.1 Preparation of chemically competent cells 59 2.3.2 Transformation of chemically competent cells 59 2.3.3 Cloning and site-directed mutagenesis 60 2.3.4 Determination of DNA concentration 61 2.4 Protein Expression and Purification 61 2.4.1 Transfection and purification of glycoproteins 61 2.4.2 SDS-PAGE and western blot 63 2.4.3 Determination of protein concentration 65 2.4.4 Silver stain of SDS-PAGE gels 65 2.4.5 Purification of sGP from ZEBOV-infected supernatant 66 2.5 Analysis of Intracellular and Surface Expression of GP1,2 66 2.6 Mass Spectrometry 67 2.6.1 Peptide mapping and disulphide bond assignment 68 2.6.2 In gel digestion of SDS-PAGE purified sGP 69 2.6.3 Data analysis 70 2.7 Barrier Function Analysis of Endothelial Cells 71 2.7.1 Preparation of chambers for impedance spectroscopy 74 2.7.2 Shear stress and impedance spectroscopy 74 2.7.3 Infection of HUVECs with Lentivirus vector expressing sGP 77 2.8 Viral Protein Database Search for C-mannosylation Motifs 77 2.9 Infectious Virus-Like Particle (iVLP) Assay 78 3. RESULTS 3.1 Structural Analysis of sGP 82 3.1.1 Construction, production and biochemical characterization of sGP 82 and cysteine mutants 3.1.2 Confirmation of primary structure and disulphide bond assignment 84 in sGP 3.1.3 Determination of site specific N-glycosylation of sGP 92 3.1.4 Identification of C-mannosylation, a novel post-translational 94 modification, on sGP 3.1.5 Alteration of the WXXW motif prevents C-mannosylation of Ebola 98 virus sGP 3.1.6 C-mannosylation of sGP in multiple cell lines and during ZEBOV 100 infection 3.2 Effect of sGP on Endothelial Barrier Function 103 3.2.1 sGP treatment of endothelial cell exposed to shear stress does not 103 affect shear stress-induced changes to barrier function 3.2.2 Endogenous expression of sGP in endothelial cells exposed to shear 105 stress does not alter shear stress-induced changes to barrier function 3.2.3 sGP and TNF-α treatment of endothelial cell exposed to shear 107 stress does not significantly affect shear stress-induced changes to barrier function 3.2.4 sGP cysteine mutants have significantly reduced abilities to rescue 107 endothelial barrier function following TNF-α treatment under static vi conditions 3.2.5 Elimination of C-mannosylation on sGP does not affect its ability to 108 rescue endothelial barrier function following TNF-α treatment 3.3 The Potential for C-mannosylation of Other Viral Proteins 111 3.3.1 Search for WXXW motifs in viral proteins that contain signal 111 peptides 3.3.2 C-mannosylation motifs are present in GP1 and GP2 114 3.3.3 Analysis of potential C-mannosylation sites on GP1,2 114 3.4 Functional Analysis of C-mannosylation Motifs in GP1,2 119 3.4.1 Expression of GP1,2 mutants was not significantly different 119 3.4.2 Mutations of tryptophans in GP1,2 alters reporter activity in an 121 infectious virus-like particle assay 3.4.3 Transport of mutant GP1,2 to the surface is not affected 122 3.4.4 Incorporation of tryptophan mutant GP1,2 constructs 129 4. DISCUSSION 4.1 Structural Analysis of sGP 132 4.1.1 Orientation of the sGP homodimer 132 4.1.2 Identification of co- and post- translational modifications 136 4.1.3 Confirmation of C-mannosylation of sGP 138 4.2 Functional Analysis of sGP on Endothelial Barrier Function 142 4.2.1 Effect of sGP on endothelial barrier function 142 4.2.2 sGP intermolecular disulphide bonds are important for its anti- 147 inflammatory effect 4.2.3 C-mannosylation of sGP is not essential for barrier function rescue 149 4.3 Potential for C-mannosylation of Other Viral Proteins 151 4.4.C-mannosylation of GP1 and GP2 152 4.5 Functional Consequences of Altering Tryptophan Residues in GP1 and 154 GP2 4.5.1 Functional importance of tryptophan residues in GP2 MPER 155 4.5.2 C-mannosylation of GP1 is not critical for GP function 158 4.6 Summary 160 4.7 Future directions 162 5.