Characterization of Orthoreovirus Core Proteins By sequencing and Proteomic Analysis of Avian Reovirus p Proteins and Development of cr-Mammalian Reovirus Core Protein Monoclonal Antibodies By Lindsay Noad A Thesis Submitted to the Faculty of Graduate Studies ln Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Medical Microbiology and Infectious Diseases University of Manitoba 'Winnipeg, Manitoba Canada olindsay Noad, October 2004 THE I]NIVERSITY OF MANITOBA FACI]LTY OF GRADUATE STT]DIES ***** COPYRIGHT PERMISSION Characterization of Orthoreovirus Core Proteins By Sequencing and Proteomic Analysis of Avian Reovirus ¡.tProteins and Development of q-Mammalian Reovirus Core Protein Monoclonal Antibodies BY Lindsay Noad A ThesisÆracticum submitted to the Faculty of Graduate Studies of The Universify of Manitoba in partial fulfillment of the requirement of the degree of Master of Science Lindsay Noad @ 2004 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 MicrofiIms 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 ovvner solely for the purpose ofprivate study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. Table of Contents TABLE OF CONTENTS I ACKNOV/LEDGEMENTS V LIST OF FIGURES VI LIST OF TABLES VM LIST OF ABBREVIATIONS IX ABSTRACT 1 a 1. INTRODUCTION J 1.1 Reovirus J 1.2 Reovirus Structure 4 1.3 Reovirus Life Cycle 7 L4 MRV vs. ARV: Similarities and Differences T2 1.5 The MRV ¡r Proteins T3 1.6 Reovirus Core Proteins T6 1.7 Antibody General Overview 20 1.8 AntibodySpecificityldentification 22 1.9 Charactenzation of Reovirus Antibodies 25 1.10 Goals of This Research 26 2. MATERIALS AND METHODS 28 2.T Cells and Viruses 28 2.2 Antibodies 29 2.3 Virus Manipulation 32 2.3.1 Viral Passage 32 2.3.2 Virus Plaque Assay JJ 2.3.3 Mammalian Virus Purification 34 2.3.4 Purification of ARV138 34 2.3.5 Preparation of Mammalian Reovirus Cores 35 2.4 Sequencing the ARV 138 and ARV 176 Ml, M2 35 and M3 genes 2.4.1 Preparation of viral template 35 2.4.2 Primer Design 36 2.4.3 Reverse transcription polyn,erase chain 36 reaction (RT-PCR) 2.4.4 3' Ligation for ARV Gene-End Sequencing 38 2.4.5 Cycle sequencing reactions 39 2.4.6 Sequence Analysis 40 2.5 Proteomic Analysis of Avian Reovirus Proteins 40 2.5.1 Protein Assay 40 2.5.2 Tris-Glycine-Urea SDS-PAGE of Avian 4I Reovirus Proteins 2.5.3 In-Gel Digestion 42 2.5.4 Mass spectrometric analysis 43 2.6 Characterization of Reovirus Antibodies 44 2.6.T ELISA 44 2.6.2 Western Blots 44 II 2.6.3 Electro-Elution of Reovirus À proteins from 45 TGU Gels 2.6.4 Antibody Column preparation 46 2.6.5 Immunoprecipitation Linked to Mass spectrometry 47 2.6.6 Immunoprecipitation Linked to SDS_pAGE 4g 3. RESULTS 49 3.1 ARV M Gene Sequence Analysis 49 3.1.1 M1 gene and pA protein sequence of ARVl3g 50 and ARV176 3.1.2 M2 gene and pB protein sequence of ARV13g 50 and ARV176 3.I.3 M3 gene sequences and ¡rNS sequence of ARVl3g 55 and ARV176 3.2 ARV Predicted p Protein Sequence Analysis 57 3.2.1 Comparisons of the ¡rA protein sequences 57 3.2.2 Comparisons of the ¡rB protein sequences 66 3.2.3 Comparisons of the pNS protein sequences 74 3.2.4 Mass spectrometric analysis of the ARV ¡"rA g5 and pB proteins 3.3 Charactenzation of g-Reovirus Core Monoclonal gg Antibodies 3.3.I cr-Reovirus Core Monoclonal Antibodies gg m 3.3.2 Production of cr-Reovirus Core 9l Monoclonal Antibodies 3.3.3 Charactenzation of a-Reovirus Core 91 Monoclonal Antibodies by Western Blot 3.3.4 Charactenzation of cr-Reovirus Core Monoclonal 95 Antibodies by TGU Gel, Protein Elution and Westem Blot 3.3.5 Charactenzation of c¿-Reovirus Core Monoclonal 100 Antibodies by Immunoprecipitation Linked to Mass Spectrometry 3.3.6 Charactenzation of c¿-Reovirus Monoclonal 101 Antibody Specificity by Immunoprecipitation Linked to SDS-PAGE and Mass Spectrometry 4. DISCUSSION 109 4.I Sequencing of the ARV M Genes and ¡r Proteins 109 4.2 Charactenzation of Reovirus Core Specific Monoclonal 113 Antibodies 4.3 Conclusions 119 4.4 Future Directions l2l 5. REFERENCES T24 IV Acknowledgments I would like to thank my committee members, Dr. John Wilkins and Dr. Heinz Feldmann for their invaluable advice, ideas and always listening and offering help when I needed it. I would especially like to thank Dr. Wilkins for opening his laboratory and office up to me and always having an open door when I needed it. I would also like to thank the members of the Coombs' lab, who have provided support, friendship and advice throughout the course of my project. These include Trina Racine, Ita HadZisej dió, Laura Hermann, Anh Tran, Wanhong Xu, Jieyuan Jiang, paul Hazelton, Chris Robertson and Chris Lindquist. I would like to thank the other graduate students in the department who always make the day interesting. My appreciation as well to the members of my "other lab", the RDU lab. I would especially like to thank Dr. Tracey Weiler, who took me under her wing, taught me more than I could imagine about immunology and everything I know about organization and still managed to be a great friend. Thanks also to Patti Sauder, Sandra Sousa and Keding Cheng for the wide variety of guidance and support they have given me over the last two years. I would like to thank my family and füends for their love and support through this whole process. I could not have made it this far without them. Most importanfly,I would like to acknowledge and express my gratitude to my supervisor, Dr. Kevin Coombs. He has taught me so much and has helped me become a better person by always being there, no matter what the situation, with his trusted advice and words of wisdom. All of his patience, guidance and humour during my time in the Coombs lab are greatly appreciated. List of Figures 1. Coding assignments and locations of structural proteins 5 within reovirus particles 2. Mammalian Reovirus Life Cycle I 3. The MALDI QqTOF mass spectrometer ¿) 4. General Overview of the Production of Monoclonal Antibodies 30 5. Sequences of the 5' and 3'non-translated regions of ARVl38 s3 and ARV176 6. Alignment of the deduced ¡rA amino acid sequences of ARV138 60 and ARV176 7. Phylogenetic Tree Analyses of the prototype ARV deduced 62 ¡rA proteins and homologous proteins in other reoviruses, with homology as determined by BLAST analysis 8. Secondary structure predictions of pA proteins 64 9. Alignment of the deduced ¡rB amino acid sequences of 68 ARV138 and ARV176 10, Phylogenetic Tree Analyses of the prototype ARV deduced 70 pB proteins and homologous proteins in other reoviruses, with homology as determined by BLAST analysis 11. Secondary structure predictions of ¡rB proteins 72 t2. Alignment of the deduced ¡rNS amino acid sequences of 75 ARV138 and ARV176 13. Phylogenetic Tree Analyses of the prototype ARV deduced 80 ¡rNS proteins and homologous proteins in other reoviruses, with homology as determined by BLAST analysis T4, Secondary structure predictions of ¡rNS proteins 82 15. Mass spectrometric analyses of gel-purified ARV138 ¡rB protein 86 T6, Mass spectrometric analyses of gel-purified ARV138 ¡rA protein 89 VI 17. V/estem Blot Analysis of Reovirus Core Specific Monoclonal 93 Antibodies 18. Tris-Glycine-Urea Gel Separation of Reovirus Core Proteins 96 19. Western Blot Analysis of Reovirus Core Specific Monoclonal 98 Antibodies #2-3Cl I and #2-5C2 20. Charactenzation of Reovirus Core Specific Monoclonal t02 Antibody # 1 -3C 1 1 by Immunoprecipitation 2 1 . Mass spectrum of )" 1 pulled down by Reovirus Core Specific 10s Monoclonal Antibody #1 -3C 1 1 22.Mass spectrum of o2 pulled down by Reovirus Core Specific r07 Monoclonal Antibody #1 -3C 1 1 VII List of Tables 1. Properties of Mammalian Reovirus Proteins 14 2. Avian Reovirus Primers for Amplification and 37 Sequencing of the M Genes 3. Features of ARV 138 and ARV 176 M Genes and p Proteins 51 4. Pairwise Comparisons of ARV 138 and ARV 176 pA proteins 52 with Mammalian Reovirus p2 proteins 5. Pairwise Comparisons of ARV 138 and ARV 176 pB proteins 56 with Mammalian Reovirus p1 proteins 6. Pairwise Comparisons of ARV 138 and ARV 176 ¡rNS proteins 58 with Mammalian Reovirus ¡iNS proteins 7. Charactenzation of Reovirus Core Specific Monocional 92 Antibodies VM List of Abbreviations a.a. amino acids ACN acetonitrile ARV avian reovirus ATPase adenosine triphosphatase BCA bicinchoninic acid BRV baboon reovirus BSA bovine serum albumin cDNA complementary deoxyribonucleic acid CPE cytopathic effect dATP deoxyadenosine triphosphate DEPC diethyl pyrocarbonate DMSO dimethyl sulfoxide dNTP deoxynucleoside triphosphatase ds double-stranded DTT dithiothreitol EDTA ethylenediamine tetraacetate ELISA enzyme-linked immunosorbent assay FCS fetal calf serum HO Buffer homogenization buffer Ig immunoglobulin IgG immunoglobulin G ISVP intermediate/infectious subviral particle L929 mouse f,rbroblast cel1 line MALDI QqTOF matrix-assisted laser desorption/ionization quadrupole time of flight MEM minimal essential medium MOI multiplicity of infection mRNA messenger ribonucleic acid MRV mammalian reovirus MS mass spectrometry/spectrometer MS/À4S tandem mass spectrometry mJz mass/charge ratio NBV Nelson Bay reovirus NTPase nucleotide triphosphatase ORF open reading frame PBS phosphate buffered saline PCR polyrnerase chain reaction PFU plaque forming unit PI post infection QM5 continuous quail cell line RdRp RNA-dependent RNA polymerase RNA ribonucleic acid RNase ribonuclease RRV reptilian reovirus x RT-PCR reverse transcription polynerase chain reaction SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SS single-stranded T1L type 1 Lang T2J type 2 Jones T3D type 3 Dearing TBST Tris-Buffered Saline f Tween TFA trifluoroacetic acid TGU Gel Tris-Glycine-Urea Gel TOF time of flight VSP viable serum protein volume per volume wlv weight per volume X Abstract Reovirus is a double-stranded RNA virus wíth a segmented genome.