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UMI* SUB-CELLULAR LOCALIZATION OF THE GRAPEVINE RUPESTRIS STEMPITTING-ASSOCIATED VIRUS REPLICASE A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph By SEAN PROSSER In partial fulfillment of requirements for the degree of Master of Science November, 2009 ©Sean Prosser, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Vour Tile Votre reference ISBN: 978-0-494-58413-2 Our file Notre reference ISBN: 978-0-494-58413-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada ABSTRACT SUB-CELLULAR LOCALIZATION OF THE GRAPEVINE RUPESTRIS STEMPITTING-ASSOCIATED VIRUS REPLICASE Sean William John Prosser Advisor: University of Guelph, 2009 Dr. Baozhong Meng Co-Advisor: Dr. Peter Krell The replicase polyprotein of Grapevine rupestris stem pitting-associated virus (GRSPaV) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cells and found to form punctate structures in the cytoplasm, which were hypothesized to represent viral replication vesicles. A region within the methyltransferase (MTR) domain was found to be responsible for the formation of punctate structures that aligned along actin microfilaments, but did not associate with the ER, Golgi stacks, or peroxisomes. An arginine residue (R173) and a tryptophan residue (W184) within the MTR domain were tested as putative critical residues required for membrane association based on the formation of the observed puncta; however, replacement of these residues had no effect on punctal formation. The RNA-dependent-RNA-polymerase domain of the replicase was found to form aggregate structures in the cytoplasm when expressed alone, but co- localized with the puncta formed by the MTR during co-expression experiments, suggesting a possible interaction between these two proteins. ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Baozhong Meng, for giving me the opportunity to work in his lab, as well as for his advice and help throughout the course of my degree. I would like to thank my advisory committee, Dr. Peter Krell, who was also my co-advisor, and Dr. Paul Goodwin. I would also like to thank Dr. Jay Subramarian for providing Vitis rupestris suspension cells and callus tissue. Finally I would like to thank the current and former members of the Meng, Krell, and Mullen labs, family, and friends for their advice and support. i TABLE OF CONTENTS TABLE OF CONTENTS.. ii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS viii LIST OF VIRUS ABBREVIATIONS ix CHAPTER I: INTRODUCTION 1 1.1 Viticulture 1 1.2 Rugose Wood Complex 2 1.3 Rupestris Stem Pitting 5 1.4 Flexiviridae 6 1.4.1 Genera and Taxonomy 6 1.4.2 Physical Characteristics 6 1.4.3 Host Range 7 1.4.4 Symptoms of Infection ; 7 1.4.5 Transmission 10 1.4.6 Genome Structure 11 1.5 Positive Sense RNA Virus Replication 16 1.5.1 Positive RNA Virus Superfamilies 16 1.5.2 Replicase Expression and Processing 17 1.5.3 Host Proteins , 21 1.5.4 Site of Replication 24 1.5.5 Membrane Association 26 1.5.6 Replication of Viral RNA 30 1.6 Grapevine Rupestris Stem Pitting-associated Virus 38 1.6.1 Physical Characteristics 38 1.6.2 Molecular Characteristics 38 1.6.3 ORF1: The viral replicase 41 1.6.4 ORF2, ORF3, and ORF4: The viral movement proteins 42 1.6.5 ORF5: The viral capsid protein 43 n 1.6.6 Symptoms and Transmission.. 44 1.6.7 Population Structure , 45 1.7. Hypotheses 46 CHAPTER 2: MATERIALS AND METHODS 48 2.1 Polymerase Chain Reaction (PCR) 48 2.2 Restriction Digestions 48 2.3 PCR Purification and Gel Extraction of DNA 49 2.4 De-phosphorylation, Ligation, and Transformation of DNA 49 2.5 Generation of Chemically Competent E. coli 51 2.6 Generation and Transformation of Electrocompetent.fi'. coli 51 2.7 Tobacco Bright Yellow (BY-2) Tissue Culture 52 2.8 Isolation of Tobacco BY-2 Protoplasts 53 2.9 Transfection of Tobacco BY-2 Protoplasts 54 2.10 Biolistic Bombardment and Immunostaining of Tobacco BY-2 Cells 55 2.10.1 Biolistic Bombardment , 55 2.10.2 Fixation and Immunostaining 58 2. 11 Epifluorescent Microscopy 59 2.11.1 Preparation of BY-2 Cells for Microscopic Examination 59 2.11.2 Preparation of BY-2 Protoplasts for Microscopic Examination 59 2.11.3 Epifluorescent Microscopy 60 2.12 Construction of Plant Expression Constructs 61 2.12.1 Full-length Replicase (pRSP-REP:GFP) 61 2.12.2 Truncation Constructs (pMTRASD:GFP, pMTR:GFP, pMTR:mRFP, pSD:GFP, pPOL:GFP) 66 2.12.3 Construction of pMTR:GFP (R173A), pMTR:GFP (W184A), and pMTR:GFP(R173A,W184A) 72 2.12.4 Construction of pER-mRFP 73 CHAPTER 3: RESULTS 75 3.1 The GRSPaV replicase forms punctate structures in the cytoplasm of tobacco BY-2 cells and protoplasts 75 3.2 A sub-domain within the MTR domain is important for the formation of the viral puncta 81 iii 3.3 The GRSPaV replicase polyprotein residues R173 and W184 are not critical for the formation of the viral puncta 90 3.4 The viral puncta appear to localize to actin microfilaments and adjacent to the ER lumen 91 3.5 The RdRp domain of the GRSPaV replicase forms aggregate structures in the cytoplasm when expressed alone 103 CHAPTER 4: DISCUSSION AND CONCLUSIONS 106 4.1 The GRSPaV replicase forms punctate structures when expressed in tobacco BY-2 cells and protoplasts 106 4.2 Amino acid residues 132-207 of the MTR domain of the GRSPaV replicase are responsible for the formation of viral puncta 107 4.3 The GRSPaV replicase polyprotein residues R173 and W184 are not critical for the formation of viral puncta 109 4.4 The viral puncta localize adjacent to the ER lumen and appear to align along actin microfilaments 113 4.5 The GRSPaV RdRp domain forms aggregate structures in tobacco BY-2 protoplasts when expressed alone and may interact with the MTR domain 115 4.6 Conclusions : 117 REFERENCES 123 APPENDIX I: INSTABILITY OF pREP:GFP IN E. coli DH5a 131 APPENDIX II: GENERATION OF GRAPEVINE PROTOPLASTS 141 APPENDIX III: IMMUNOSTAINING OF TOBACCO BY-2 PROTOPLASTS 145 IV LIST OF TABLES Table Page Table 1: Primers used in this study. 62 v LIST OF FIGURES Figure Page Figure 1: Symptoms of the four diseases comprising the Rugose Wood Complex. 3 Figure 2: Electron micrograph images showing virions of six representative members of the family Flexiviridae. 8 Figure 3: Genome structure of five representative members of the family Flexiviridae. 12 Figure 4: Replicase structures of the four positive RNA virus superfamilies. 18 Figure 5: Three dimensional structure of the Semliki Forest virus monotopic membrane association signal. 28 Figure 6: Replication of a positive RNA viral genome utilizing sub-genomic RNA production. 31 Figure 7: Physical and molecular structure of Grapevine rupestris stem pitting-associated virus. 39 Figure 8: Vector map of pRTL2. 67 Figure 9: Full-length and truncated replicase constructs used in this study. 76 Figure 10: Epifluorescent images of tobacco BY-2 cells and protoplasts expressing the full-length GRSPaV replicase. 78 Figure 11: Predicted amphipathic regions of the GRSPaV methyltransferase domain. 82 Figure 12: Nucleotide alignment of the GRSPaV methyltransferase domain with the Semliki Forest virus nsPl replication protein and its relative position to a predicted amphipathic region within the "sub-domain". 85 Figure 13: Epifluorescent images of tobacco BY-2 protoplasts expressing regions of the methyltransferase domain. 88 Figure 14: Mutant truncation constructs used in this study. 92 Figure 15: Epifluorescent images of tobacco BY-2 protoplasts expressing mutant truncation constructs. 94 vi Figure 16: Epifluorescent images of tobacco BY-2 protoplasts co-expressing the methyltransferase domain with an ER marker protein. 97 Figure 17: Epifluorescent images of tobacco BY-2 protoplasts co-expressing the methyltransferase domain with Golgi and peroxisome marker proteins. 99 Figure 18: Epifluorescent images of tobacco BY-2 protoplasts co-expressing the methyltransferase domain with an actin marker protein.
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