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Genome Sequence and Molecular GENOME SEQUENCE AND MOLECULAR CHARACTERIZATION OF Homalodisca coagulata virus-1, A NOVEL VIRUS DISCOVERED IN THE GLASSY- WINGED SHARPSHOOTER (HEMIPTERA: CICADELLIDAE) By LAURA E. HUNNICUTT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Laura E. Hunnicutt ACKNOWLEDGMENTS There are many people without whom effecting this thesis and the research described herein would not have been possible. I would first like to take the opportunity to thank my supervisory chair, Dr. Ron Cave. Dr. Cave was instrumental not only as an excellent instructor but also as a constant source of direction, support, and encouragement over the past two years. I would also like to thank the supplementary members of my committee including Dr. Chuck Powell for his approachability and insight regarding my thesis research as well as his adept critical review of the resultant journal manuscripts. I would like to extend a special thanks to Dr. Wayne Hunter who generously apportioned CRIS funds to finance this project and exposed me to a remarkably creative way of viewing science and scientific discovery. I am very appreciative to my colleagues at the USDA ARS U.S. Horticultural Research Laboratory (Ft. Pierce, Florida) for providing an educational, and often entertaining, work environment. I would like to thank Matt Hentz for help with insect collection, Dr. Phat Dang for help with EST sequencing, Dr. Bob Shatters for helpful discussions pertaining to general molecular biology concepts/theorems, and Dr. Laura Boykin for providing critical assistance with phylogenetic analyses on this and other works. I would especially like to thank Jerry Mozoruk, with whom I spent nearly all of graduate school working side-by-side. I will always be grateful to him for being such a wonderful friend and mentor to me. iii I would also like to acknowledge those associates who acted as outside collaborators. I thank Drs. Chris Tipping and Russ Mizell (University of Florida, NFREC) for help in collecting Homalodisca coagulata and Oncometopia nigricans in Quincy, Florida, and their direction regarding collections made in Cairo, Georgia. I am thankful to Dr. Heather Costa (University of California, Riverside) as well as Dr. Gary Puterka and Mike Reinke (USDA ARS AFRS) for samples of H. coagulata from California; Tina Winstrom (University of California, Berkeley) and Robert Keiffer (University of California, Davis Hopland Field Station) for samples of Draeculacephala minerva and Graphocephala atropunctata from California; and Dr. Renato Bautista (Hawaii Department of Agriculture Plant Industry Division) for valuable information regarding H. coagulata collection sites in Oahu, Hawaii. Finally, I am very grateful to my family for their unwavering support and encouragement. I would especially like to thank my parents, Kermit Hunnicutt and Barbara Hunnicutt-Greenwell, for instilling in me the work ethic and ambition which enabled me to reach this goal. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION ........................................................................................................1 2 MATERIALS AND METHODS .................................................................................4 Sequencing of the HoCV-1 Genome.............................................................................4 cDNA Library Construction..................................................................................4 5′-Rapid Amplification of cDNA Ends (RACE)...................................................5 Sequence Verification and Cloning..............................................................................6 Computer Analysis of HoCV-1 Nucleic Acid and Deduced Protein Sequences..........7 Theoretical Modeling of the HoCV-1 RNA-dependent RNA Polymerase (RdRp)......8 Development and Application of an RT-PCR Assay to Determine the Geographical Distribution and Natural Host Range of HoCV-1 .............................8 Insect Sampling .....................................................................................................8 Sample Preparation and RT-PCR Analysis...........................................................9 RT-PCR Primer Design.......................................................................................10 Determination of the Sensitivity of Amplification of HoCV-1 by RT-PCR .......10 3 RESULTS AND DISCUSSION.................................................................................12 Nucleotide Sequence of HoCV-1................................................................................12 Similarity with Other Taxa in Regards to Partition and Arrangement of the Genome..................................................................................................................20 Alignment of the Amino Acid Sequences of Viral Non-structural Proteins with HoCV-1 ORF1 .......................................................................................................21 The RNA-dependent RNA Polymerase (RdRp) Domain....................................21 The Nucleotide-binding (Helicase) Domain .......................................................26 The Protease Domain ..........................................................................................29 The Intergenic Region (IGR) of HoCV-1 ...................................................................30 v Mapping of the Coding Region of the Structural Proteins .........................................32 Analysis of the HoCV-1 ORF2 Amino Acid Product .........................................32 Structural Models of the HoCV-1 Capsid Proteins..............................................35 Phylogenetic Analysis ................................................................................................38 Development of an RT-PCR Assay as a Diagnostic Tool for Sharpshooter Virus Detection................................................................................................................39 Incidence of HoCV-1 Infection in Geographically Disparate Regions of North America..................................................................................................................41 4 CONCLUSION...........................................................................................................44 APPENDIX -- RT-PCR ASSAY GEL IMAGES..............................................................46 LIST OF REFERENCES...................................................................................................50 BIOGRAPHICAL SKETCH .............................................................................................58 vi TABLE Table page 1 Survey of sharpshooter populations for the presence of HoCV-1............................42 vii LIST OF FIGURES Figure page 1 The nucleotide sequence of HoCV-1 genomic RNA................................................12 2 Restriction enzyme analyses of a cloned cDNA spanning the complete HoCV-1 genome .....................................................................................................................18 3 Multiple sequence alignment of the putative RNA-dependent RNA polymerase (RdRp) domain of suggested members of the genus Cripavirus .............................22 4 Structural model analysis of the HoCV-1 RNA-dependent RNA polymerase (RdRp)......................................................................................................................23 5 Structure of the HoCV-1 RNA-dependent RNA polymerase (RdRp) showing conserved motifs relevant to enzymatic activity......................................................25 6 Multiple sequence alignment of the nucleotide binding (helicase) domain of suggested members of the genus Cripavirus............................................................27 7 Multiple sequence alignment of the protease domain of suggested members of the genus Cripavirus. ...............................................................................................29 8 Multiple nucleotide sequence alignment of the intergenic regions (IGR) of CrPV, PSIV, RhPV, and HoCV-1 .............................................................................30 9 Secondary structure of the HoCV-1 internal ribosomal entry site (IRES) within the intergenic region as predicted by Mfold.............................................................31 10 Multiple alignment of conserved amino acid sequences specific for capsid protein 2 (CP2), 3 (CP3) and 1 (CP1) of suggested members of the genus Cripavirus.................................................................................................................33 11 Organization of the genome of HoCV-1, with the structural proteins highlighted to show the order of the individual capsid proteins .................................................34 12 The structures of the four HoCV-1 capsid proteins, CP1-4......................................36 13 Phylogenetic analysis
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