/ THE CHARACTERISATION OF ORNITHOGALUM MOSAIC VIRUS Johan Theodorus Burger University of Cape Town A dissertation submitted in fulfilment of the requiremements for the Degree of Doctor of Philosophy in the Faculty of Science, University of Cape Town. Cape Town, November, 1990 ·F-~~-=-~:.r:-":':."·.-:_~-~·;-.~--:-~·_::--:"_-.:-:::·_, ·_-:-... _... ';' _-:_: = -~-- -·-:--~ -~u 7 1! ~h: ~;;;-:f)~;_i~y :~(.\ ~~ ~~'·:~~ .. ~;.;s-~::;3:,_~:~:~~ '] c~v~1 ~:.:~.- -· . ·' -:~. ;: The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town CERTIFICATION OF SUPERVISOR' In terms of paragraph GP 8 in " General rules for the Degree of Doctor of Philosophy (PhD)", I as supervisor of the candidate, Johan Theodorus Burger, certify that I approve of- the incorporation in this dissertation of material that has already been submitted or accepted for publication. Associated Professor M. B. Von Wechmar Department of Microbiology University of Cape Town i CONTENTS ACKNOWLEDGEMENTS .................................... · ii ABBREVIATIONS . iii LIST OF FIGURES . vi LIST OFTABLES ................................ ; . ix ABSTRACT............................................ X CHAPTER 1: Introduction .......... "'· . 1 CHAPTER 2: The purification and physicochemical characterisation of ornithogalum mosaic virus. 33 CHAPTER 3: Serology of ornithogalum mosaic virus . 48 CHAPTER 4: Biological aspects of ornithogalum mosaic virus . 70 CHAPTER 5: Molecular cloning and nucleotide sequencing of ornithogalum mosaic virus. 88 CHAPTER 6: The expression of ornithogalum mosaic virus coat protein in E. coli. 113 CHAPTER 7: General discussion and conclusions . 127 APPENDIX A: Virus names and acronyms . 130 APPENDIX B: Standard methods . 134 REFERENCES CITED . 155 ii ACKNOWLEDGEMENTS I wish to sincerely thank my supervisors; Associated Professor Barbara von Wechmar for her advice and endless enthusiasm throughout this project, and Dr. Edward Rybicki for his invaluable advice and encouragement. Also for the tireless rea4ing of manuscripts. I am grateful to Dr. J. T. Meynhardt, Director of the Vegetable and Ornamental Plant Research Institute, for the opportunity to do this project on a full-time basis at UCT, also for his patience towards the end. A special word of thanks to Mr. G. Kasdorf for his expert assistance with electron microscopy and photography. I wish to thank the technical staff of the Microbiology Department, especially Mr. J. Hamman for virus purifications, Mr. D. Solomons for plant maintenance, Messrs. D. Roussouw and W. Jacobs for antiserum preparation, and also Cde. Peter Buckton for general assistance and for sha_ring his daily newspaper with me for the past few years. Many thanks to my good friend and colleague Reon Brand for many shared hours, in the lab and elsewhere. Finally, I wish to thank my parents; my father for initially providing the challenge, and them both for their continual support and encouragement, also when times were tough. I acknowledge the financial support of the Vegetable and Ornamental Plant Research Institute, Department of Agricultural Development. .. iii ABBREVIATIONS A adenine I adenosine AI amorphous inclusion APS ammonium persulphate ATP adenosine 5'-triphosphate 13-gal beta-galactosidase BCIP 5-bromo-4-chloro-3-indolyl phosphate bis N,N'-methylene-bisacrylamide bp base pair BSA bovine serum albumin c cytidine I cytosine Ci curie CI cytoplasmic inclusion CIP calf intestinal alkaline phosphatase CP coat protein cpm counts per minute Da dalton DA5-ELISA double antibody sandwich enzyme-linked immunosorbent assay dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate ddATP dideoxyadenosine triphosphate ddCTP dideoxycytidine triphosphate ddGTP dideoxyguanosine triphosphate ddNTP dideoxyn ucleoside triphosphate ddTTP dideoxythymidine triphosphate DEAE diethy laminoethy 1 DEPC dieth y 1pyrocarbona te dGTP deoxyguanosine triphosphate DMSO dimethyl sulphoxide iv DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP deoxynucleoside triphosphate dpni.' disintegrations per minute DTE dithioerythritol DIT dithiothreitol dTIP deoxythymidine triphosphate EDTA ethylenediaminetetraacetic acid g gravitational acceleration G guanine I guanosine HC helper component HPLC high performance liquid. chromatography IEB immunoelectroblotting · IgG gamma-immunoglobulin ISEM immunosorbent electron microscopy Kb kilobase MAb monoclonal antibody MOPS morpholinepropanesulphonic acid Mr relative molecular weight NBT nitro-blue tetrazolium chloride NI nuclear inclusion OD optical density oligo(dTI oligodeoxythymidylic acid ORF open reading frame PBS phosphate-buffered saline PEG polyethylene glycol poly(A) polyadenylic acid PVP polyvinylpyrrolidone RNA ribonucleic acid RNase ribonuclease SD5-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis spp. species T . thymine I thymidine v TEMED N,N,N',N'-tetramethyl-ethylenediamine Tris tris(hydroxymethyl)aminomethane u unit uv ultraviolet v/v volume per volume w/v weight per volume X-gal 5-bromo-4-chloro-3-in dol y 1-P-D-galactosidase vi LIST OF FIGURES Fig. 1.1. The genetic map of tobacco etch virus. ..... 23 Fig. 1.2. Polyprotein processing of tobacco etch virus............. 26 Fig. 1.3. Map of the areas where most South African Ornithogalum spp. grow naturally............................ 28 Fig. 1.4. An ornithogalum cultivar produced in the VOPRI breeding program. 29 Fig. 1.5. Map of the areas where Lachenalia spp. grow naturally...... 31 Fig. 1.6. A lachenalia cultivar from the VOPRI breeding programme.... 32 Fig. 2.1. Purified preparation of OMV stained with 2% (w /v) ammonium molybdate.......................... 42 Fig. 2.2. ISEM of OMV particles with antiserum directed against a Dutch isolate of OMV.......................... 42 Fig. 2.3. Particle length distribution of OMV.................. 43 Fig. 2.4. SD5-PAGE ofOMV CP.......................... 44 Fig. 2.5. Molecular weight determination of OMV RNA in formaldehyde agarose gels ........................ 44 Fig. 2.6. Elution profile of OMV RNA in oligo(dT)-cellulose chromatography.............................. 45 Fig. 3.1. Excision of bulb scale tissue sections without damaging growth points. ... 58 Fig. 3.2. Direct DA5-ELISA showing the serological interrelationships among South African isolates of OMV......... :....... 59 Fig. 3.3. Immunoelectroblot of three South African isolates of OMV.... 59 Fig. 3.4. lmmunoelectroblot probed with different potyvirus antisera... 61 vii Fig. 3.5. DOT-ELISA of the leaf extracts of young ornithogalum and lachenalia plantlets ............................. 65 Fig. 4.1. Localities from where ornithogalum and lachenalia leaf samples were collected ......................... ~ . 76 Fig. 4.2. Ornithogalum mosaic virus symptoms on ornithogalum leaves. 77 Fig. 4.3. Ornithogalum mosaic virus symptoms on ornithogalum flower stalks. 78 Fig. 4.4. Flower stunting and deformation in ornithogalum caused by severe OMV infection .................... 79 Fig. 4.5. The unknown icosahedral virus which infected ornithogalum and lachenalia. .... 86 Fig. 5.1. eDNA cloning strategy for pOM16 ................... 92 Fig. 5.2. Subcloning strategy for p0M21. .................... 94 Fig. 5.3. Colony hybridisation of OMV eDNA clones .......... · ... 97 Fig. 5.4. OMV cON A clone sizes .......................... 98 Fig. 5.5. Southern blot hybridisation of eighteen OMV clones ....... 98 Fig. 5.6. pOM16 digested with the pUC18/pUC19 polylinker restriction enzymes. 99 Fig. 5.7. Exonuclease III/S1 nuclease shortening of pOM16 and pOM21. 100 Fig. 5.8. OMV clone orientation and sequencing strategy.......... 101 Fig. 5.9. Nucleotide and deduced amino acid sequence of the OMV virion-sense eDNA ......................... 102 Fig. 5.10. The single large ORF in which the OMV sequence translated. 106 Fig. 5.11. Alignment of the deduced coat protein amino acid sequences of OMV and 14 other potyviruses ............ 109 Fig. 5.12. Phylogenetic tree calculated for potyvirus coat proteins ..... 111 viii Fig. 6.1. Subcloning of OMV coat protein in t1:1e pUEX2 expression vector. 117 Fig. 6.2. Colony hybridisation of pUEX clones ................. 120 Fig. 6.3. Immuno-colony blots of pUEX2 clones ................ 121 Fig. 6.4. SDS-PAGE of partially-purified protein extracts from E. coli containing different plasmids................. 122 Fig. 6.5. Immunoelectroblot of partially purified E. coli protein extracts .. 123 Fig. 6.6. Immunoelectroblot with a potyviru~ CP-specific monoclonal antibody (PTY-1) ...................... 124 Fig. 6.7. IEB with antisera to purified OMV and expressed ~-gal::OMV CP fusion protein ...................... 124 ix LIST OF TABLES Table 1.1 Some plant virus pathogens of ornamental bulbs .......... 5 Table 3.1 Virus isolates used in serological relationship studies ....... 53 Table 3.2 Antisera used in serological relationship studies . ..... 54 Table 3.3 Serological relationships between OMV and other potyviruses. 62 Table 3.4 DAS-ELISA of bulb scale tissue and growth points ......... 63 Table 3.5 DOT-ELISA of bulb scale tissue and growth points ......... 64 Table 3.6 DAS-ELISA of leaf extracts ......................... 64 Table 4.1 OMV transmission to potential propagation hosts ......... 81 Table