University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2009-01-01 The Role of RNA Secondary Structure in Replication of Nodamura Virus RNA2 John Howard Upton University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Virology Commons Recommended Citation Upton, John Howard, "The Role of RNA Secondary Structure in Replication of Nodamura Virus RNA2" (2009). Open Access Theses & Dissertations. 372. https://digitalcommons.utep.edu/open_etd/372 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. THE ROLE OF RNA SECONDARY STRUCTURE IN REPLICATION OF NODAMURA VIRUS RNA2 JOHN H. UPTON, III Department of Biological Sciences APPROVED: ____________________________________ Kyle L. Johnson, Ph.D., Chair ____________________________________ Germán Rosas-Acosta, Ph.D. ____________________________________ Ming-Ying Leung, Ph.D. _______________________ Patricia D. Witherspoon, Ph.D. Dean of the Graduate School Copyright © By John H. Upton, III 2009 DEDICATION This thesis is dedicated to my family and friends who have encouraged me through the years. Without their patience, understanding, support, and most of all love, the completion of this work would not have been possible. THE ROLE OF RNA SECONDARY STRUCTURE IN REPLICATION OF NODAMURA VIRUS RNA2 By JOHN H. UPTON III, B.S. THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Biological Sciences THE UNIVERSITY OF TEXAS AT EL PASO August 2009 ACKNOWLEDGEMENTS I express my gratitude and love to my wife, Janeen, and our three children for their continued support and encouragement. She never stopped believing in me, always reminding me that I could achieve anything I set my mind to. You gave me immense support, desire to be a role model to our children, and the drive to complete my tasks without pressure. Thank you. To the Command and the members of the 7220th Medical Support Unit, thank you for the support and understanding while I worked on this project. My military mentors gave me more than they realize. The person I am today is a result of all the great individuals that I have encountered throughout my tours: Leaders, Peers, and subordinates alike. I would like to convey my appreciation to Dr. Kathy Hanley and her laboratory staff, particularly Tammy Romero, for guiding me through the nuclease mapping procedures. Without you, I would not be where I am today. I am certain that without your support this project would not be done. Thank you for your confidence in me and for helping me achieve what seemed impossible. A special thanks to all of the UTEP Biology staff members who assisted me while using unfamiliar laboratory equipment: Lani Alcazar in the Biomolecule Analysis Core facility, Berenice Arriaga in the HHMI Student Molecular Research Lab Core facility, and especially Omar Hernandez in the DNA Analysis Core facility whose patience and assistance with the optimization of this project’s sequencing and mapping gels was greatly appreciated. v I would also like to acknowledge some of the outstanding faculty members at UTEP whose dedication and influence furthered my love of the sciences: Thank you, Dr. Stephen Aley for your recommendation letters and guidance throughout my time at UTEP. Thank you, Dr. Donna Ekal for your recommendations and your vital role in helping me achieve all my goals. Thank you, Dr. Tina Garza for your recommendation letters and for the wonderful experience working as your T.A. in Molecular Cellular Biology. Finally, thank you, Dr. Mahesh Narayan for your recommendations, advice, and great sense of humor. You each were an integral part of my success and have made a lasting impression. My sincere gratitude to all the members of the Johnson Lab, especially John Rosskopf, my right hand man, technical advisor and good friend for putting up with my onslaught of endless questions, volleying my ideas and concerns as they came up, and helping to keep my spirits high throughout this project. I would like to thank the members of my committee; Dr. Ming-Ying Leung for her guidance and assistance with computer modeling and predictions; Dr. German Rosas- Acosta for his advice and wisdom, which were greatly appreciated; and finally I would like to express my deep appreciation to my committee chair and mentor, Dr. Kyle L. Johnson. Your instruction on this project and beyond has helped me grow in ways I am just beginning to comprehend. Thank you for showing me that this was not an impossible task. You came through for me while I was under pressure, without hesitation. You are more of a role model to me than you know. vi ABSTRACT Our laboratory studies genome replication of positive-strand RNA viruses, using a reverse-genetic system in yeast cells. Nodamura virus (NoV) provides an excellent model system for the study of RNA replication due to its genetic simplicity, its robust yield of replication products, and its ability to replicate in a wide variety of host cells. NoV contains a bipartite positive strand RNA genome: RNA1 encodes the viral RNA replicase, while RNA2 encodes the capsid protein. The role of RNA secondary structure in the genome replication of other RNA viruses has been well established. For NoV, sequences at the 3’ end of RNA2 are critical for RNA replication and we hypothesized that the secondary structure adopted by these RNA sequences is essential for RNA replication. Three different software programs were used to generate predicted secondary structures of the 3’-terminal end of NoV RNA2; all three consistently predicted the presence of a conserved stem-loop structure within this region. We tested whether the predicted structure has biological relevance in the viral life cycle and whether its formation could be verified experimentally. The structure was deleted from a cDNA clone of RNA2 using site directed mutagenesis. Yeast cells were transformed with wildtype or mutant RNA2 together with RNA1 as a source of RdRp and RNA replication products were detected by Northern blot hybridization. Our results showed that deletion of the predicted stem-loop structure resulted in a severe defect in RNA replication. We also used nuclease mapping to confirm that NoV RNA2 forms this structure in solution. These data suggest that the 3’-terminal region plays a significant role in RNA2 replication. Its exact role is, as yet, unknown. vii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.................................................................................................v ABSTRACT.....................................................................................................................vii TABLE OF CONTENTS.................................................................................................viii LIST OF FIGURES...........................................................................................................x Chapter 1. INTRODUCTION...................................................................................................1 1.1. RNA Secondary Structure.........................................................................2 1.2. Nodaviruses……………….........................................................................5 1.3. Nodavirus RNA Replication.......................................................................7 1.4. Prediction of NoV RNA2 Secondary Structure........................................10 1.5. Summary………………………………………………................................13 2. MATERIALS AND METHODS ..........................................................................14 2.1. Cells and Growth Conditions ..................................................................14 2.2. Plasmids Used........................................................................................15 2.3. RNA Isolation and Analysis....................................................................18 2.4. Nuclease Mapping and Primer Extension Analysis................................18 3. RESULTS .........................................................................................................20 3.1. Prediction of a Stem-Loop Structure in NoV RNA2……..........................20 3.2. Effect of Stem-Loop Deletion on RNA2 Replication ..............................21 3.3. The Predicted Stem-Loop Can Form Under Biological Conditions........27 viii 4. SUMMARY AND CONCLUSIONS ............................................................................31 4.1. Summary ..............................................................................................31 4.2. Conclusions ..........................................................................................32 LIST OF REFERENCES ...............................................................................................36 CURRICULUM VITA......................................................................................................40 ix LIST OF FIGURES 1. Three types of structures adopted by RNA………………………………………………..3 2. Nodavirus genome organization schematic……………………………………………….7 3. Schematic of nodavirus RNA replication strategy………………………………………..8 4. Initiation of NoV RNA replication in transformed yeast cells…………………………….9 5. RNA Secondary Structure Predictions