REVERSE GENETIC STRATEGIES TO INTERROGATE REASSORTMENT RESTRICTION AMONG GROUP A ROTAVIRUSES BY JESSICA R. HALL A Thesis Submitted to the Graduate FACulty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in PArtiAl Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Biology August 2019 Winston-SAlem, North Carolina Approved By: SArah M. MCDonald, Ph.D., Advisor GloriA K. Muday, Ph.D., Chair DouglAs S. Lyles, Ph.D. JAmes B. PeAse, Ph.D. ACKNOWLEDGEMENTS This body of work wAs mAde possible by the influences of Countless people including, but not limited to, those named here. First, I Am thankful for my Advisor, Dr. SArah MCDonald, who paved wAy for this opportunity. Her infeCtious enthusiAsm for sCience served As A Consistent reminder to “keep getting baCk on the horse”. I Am grateful for my Committee members, Drs. DouglAs Lyles, GloriA Muday, And JAmes PeAse whose input And AdviCe Aided in my development into A CAreful sCientist who thinks CritiCAlly About her work. I Am Also thankful for Continued mentorship from my undergraduate reseArch advisor, Dr. NAthan Coussens, whose enthusiAsm for sCience inspired mine. I Am AppreCiAtive of All of my friends for their unyielding support. I Am blessed to have A solid foundation in my FAmily, both blood And Chosen. I Am thankful for my mother, LiAna HAll, who instilled in me my work ethiC And whose pride in me has inspired me to keep pursuing my dreAms. I Am forever indebted to Dr. DiAna Arnett, who has served As my personal And sCientifiC role model; thank you for believing in me, investing in me, And shaping me into the person and sCientist I am today. This thesis is dediCAted to all of you. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS.................................................................................................ii TABLE OF CONTENTS...................................................................................................iii LIST OF FIGURES AND TABLES..................................................................................vi LIST OF ABBREVIATIONS..........................................................................................viii ABSTRACT.........................................................................................................................x INTRODUCTION...............................................................................................................1 Rotavirus as a Pathogen...........................................................................................1 RV Genome and Capsid Structure............................................................................2 RV Classification.....................................................................................................6 RVA LifeCycle And Assortment of Viral RNAs......................................................10 RVA Reassortment.................................................................................................15 RVA Reverse Genetics...........................................................................................16 RVA NSP5.............................................................................................................22 Research Aims.......................................................................................................25 METHODS........................................................................................................................26 PhylogenetiC Analysis of Modern HumAn, WA-like RVA Strains..........................26 Cell Culture............................................................................................................26 Plaque Assays.........................................................................................................27 Preparation of Viral RNAs.....................................................................................28 Plasmid Construction.............................................................................................29 Generation of Recombinant RVAs using an All Plasmid Reverse Genetics System....................................................................................................................33 iii Imaging of BHK-T7 Cells Transfected with Plasmid Encoding Green Fluorescent Protein (GFP).....................................................................................34 Growth of High-Titer Viral Stocks.........................................................................34 Quantification of Viral Growth..............................................................................34 Immunoblotting......................................................................................................35 Sequencing of NSP2 And NSP5 CDNAs.................................................................36 Nucleotide Alignments...........................................................................................38 RNA Folding Analysis...........................................................................................38 Protein Alignments.................................................................................................38 RESULTS..........................................................................................................................39 The lAboratory strain P is genetiCAlly similAr to modern, Wa-like RVAs................39 WA grows to A higher titer than P in Cell Culture......................................................42 BHK-T7 Cells Are transfeCtAble And express T7 RNA polymerase..........................42 MA104 cells are permissive to RV infection..........................................................45 ModifiCAtions to the existing All plAsmid-based reverse genetiCs system yielded sucCessful resCue of wildtype rRV SA11................................................................45 Six WA genome segments were sucCessfully Cloned into reverse genetiCs veCtors....................................................................................................................50 rSA11-g11-Wa exhibits A modest repliCAtive defeCt relAtive to wildtype.............50 WA And SA11 g11 share 88% nucleotide identity...................................................53 WA and SA11 g11 +RNAs are prediCted to form different seCondary structures ................................................................................................................................55 WA And SA11 NSP5 share 87% Amino ACid identity...............................................57 iv Intergenotypic variation among NSP5 amino acid sequences lies in important functional regions.................................................................................................59 Western blot of WA And SA11 NSP5 reveAls differentiAl banding patterns At 8 h.p.i. ................................................................................................................................61 Sequencing of NSP5 and NSP2 reveAl no compensAtory mutAtions.....................63 DISCUSSION....................................................................................................................64 REFERENCES..................................................................................................................73 APPENDIX........................................................................................................................82 CIRICULUM VITAE........................................................................................................83 v LIST OF FIGURES AND TABLES Figure 1. MAture RV virion And genome Composition..........................................................5 Figure 2. HumAn RVA genotype ConstellAtions...................................................................8 Figure 3. Overview of RVA lifeCycle.................................................................................14 Figure 4. SA11 H-96 reverse genetiCs system....................................................................18 Figure 5. HumAn vs. simiAn RVA genotype ConstellAtions.................................................20 Figure 6. ImportAnt functional regions of NSP5.................................................................24 Figure 7. Cloning sCheme for reverse genetiCs Constructs ContAining WA genes.................32 Figure 8. Figure 8. Phylogeny of Archival And modern RVA VP4 genotype sequences......41 Figure 9. BHK-T7 Cells CAn uptAke And express A plAsmid encoding GFP..........................44 Figure 10. RT-PCR validation of sucCessful wildtype rSA11 RVA resCue using All plAsmid- based reverse genetiCs system............................................................................................47 Figure 11. Growth Comparison of rSA11-wt And rSA11-g11-WA during one Cycle of infeCtion.............................................................................................................................52 Figure 12. PAirwise nucleotide Alignments of WA And SA11 genome segment 11..............54 Figure 13. PrediCted seCondary structures of WA And SA11 g11 +RNAs............................56 Figure 14. PAirwise amino aCid alignments of WA and SA11 NSP5 and NSP6................58 Figure 15. IntergenotypiC variAtion among NSP5 amino aCid sequences.........................60 Figure 16. Western blot of WA and SA11 NSP5 reveAls differentiAl banding patterns.....62 Figure 17. NSP5 Amino ACid Changes And importAnt functional regions.............................70 TAble 1. RV proteins, functions, genotypes,