PARVOVIRUS CAPSID STRUCTURES, LIGAND BINDING INTERACTIONS, AND ENDOGENOUS VIRAL ELEMENTS A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy by Heather Merrick Callaway August 2018 ©2018 Heather Merrick Callaway PARVOVIRUS CAPSID STRUCTURES, LIGAND BINDING INTERACTIONS, AND ENDOGENOUS VIRAL ELEMENTS Heather Merrick Callaway, Ph.D. Cornell University 2018 Parvoviruses are among the simplest of viruses, with capsids composed of variants of a single structural protein. These capsids mediate many of the processes required for infection and are remarkably stable. However, antibody binding or mutations to the capsid can disrupt these processes and block infection. This dissertation discusses mutations to the canine parvovirus (CPV) capsid that result in loss of infection, the interaction between CPV and the transferrin receptor (TfR), and parvovirus endogenous viral elements (EVEs), which contain ancient parvovirus gene sequences. I found that point mutations to CPV VP2 residues 270, 272, 273, and 299/300 result in loss of viral infectivity. Mutation of residue 270 results in loss of a sub-molar proteolytic cleavage event in VP2 and increases capsid stability, residue 272 mutation causes loss of capsid assembly, and residue 273 mutation results in assembled capsids being trapped in the nucleus. Mutation of VP2 residues 299 and 300, which are associated with TfR binding, to lysine disrupts the interaction between CPV and the TfR, inhibiting infection but still allowing receptor binding and uptake into cells. I also found that CPV has different interactions with TfRs from different host species, binding strongly to some TfRs and very weakly to others, even though each TfR can mediate a successful infection. TfRs from different species also had varying levels of occupancy on CPV capsids, with up to 12 black-backed jackal TfRs, but only 1-2 feline TfRs binding to each capsid, and it is possible that the feline TfR induces a conformational change in CPV that inhibits binding of additional TfRs. Antibody binding could also disrupt the CPV/TfR binding interaction, suggesting a possible mechanism of virus neutralization. To examine the structure and function of ancient parvovirus capsids, I expressed the VP2 gene from three different parvovirus EVEs. VP2 expressed from an EVE in the M. spretus genome assembled into capsids, and I determined that these capsids were highly stable, could bind to N-Acetylneuraminic acid, and were endocytosed into murine cells. The results in this dissertation provide new information about parvovirus infection, receptor binding interactions, and evolution, and further the understanding of how infection occurs and may be disrupted. BIOGRAPHICAL SKETCH Heather Callaway attended White Station High School and graduated in 2009. She went to college at Emory University and graduated summa cum laude in 2013 with a double major in biology and physics. Throughout college, Heather worked in several research laboratories, with projects including doing behavioral experiments on rats, examining how hypoxia affects the growth of cancer cells, and sequencing rhodopsin and other photoreceptor genes in fish. Her undergraduate thesis work focused on understanding how cellular proliferation in Drosophila melanogaster was affected by mutations to the gene domino. Heather became interested in working with viruses after learning about bacteriophages in high school biology. Once enrolled in the Ph.D. program at Cornell University, Heather rotated in the labs of Dr. Joel Baines, Dr. John March, and Dr. Colin Parrish. Since joining Colin Parrish’s lab, Heather has been working on understanding how parvoviruses interact with receptors and antibodies, and what makes them infectious. After graduating from Cornell, Heather will do a postdoc in the lab of Dr. Erica Ollmann Saphire, working on rabies virus glycoprotein structure. iii For my parents, Dawn and Jay, and my sister, Erin iv ACKNOWLEDGMENTS Thank you to everyone who helped me throughout my time in graduate school. I am very grateful to have had Colin Parrish as a mentor. Colin has provided me with excellent feedback on my research, experimental design, and writing, and I have become a better scientist because of his guidance. To Wendy Weichert, I owe a debt of gratitude for training me on how to do all of the biochemical assays I needed for my research, for help in troubleshooting my experiments, and for excellent technical support. Thank you for keeping us sane and the lab running, and for everything else you do. I am grateful to my committee members, Dr. Linda Nicholson, Dr. Gary Whittaker, and Dr. Bettina Wagner, for their help and guidance over the past five years, particularly on experimental design. I am also very grateful to Shelagh Johnston for all the help with sorting out scheduling and travel, as well as for keeping me on my toes. I am not sure I will ever be able to look at the California highway system the same way again. To Karen, Matt, Melissa, Becky, Brynn, Ian, Brian, Esteban, Furkat, Ed, Chip, and Wayne – thank you for your friendship, for making the lab a fun place to work, and for testing out all of my culinary experiments, good and bad. Thank you to all the folks at Baker who have made this such a wonderful place to work. I owe thanks, lastly, to my parents, for everything they do, and to my sister, for keeping me sane and from falling too far down the rabbit hole into scientific jargon. I agree – tea should be steeped, not incubated. v TABLE OF CONTENTS Biographical sketch ................................................................................................................... iii Acknowledgements ................................................................................................................... v List of figures ............................................................................................................................ x List of tables ............................................................................................................................. xii CHAPTER ONE: Introduction ................................................................................................. 1 1.1 Emergence and importance of canine parvovirus ................................................... 2 1.2 Viral proteins and capsid structure ......................................................................... 3 1.3 Parvovirus receptor binding .................................................................................... 7 1.3a Canine parvovirus and the transferrin receptor ......................................... 7 1.3b Sialic acid binding.................................................................................... 10 1.4 Parvovirus infection ............................................................................................... 11 1.4a Uptake, endosomal escape, and intracellular trafficking ......................... 11 1.4b DNA uncoating, RNA splicing, and protein expression .......................... 13 1.4c Capsid assembly, genome replication, and packaging ............................. 14 1.4d Nuclear egress and release from the cell .................................................. 17 1.5 Evolution and host adaptation of canine parvovirus .............................................. 18 1.6 Parvovirus/antibody interactions ........................................................................... 20 1.6a Antibody recognition of canine parvovirus.............................................. 20 1.6b Mechanisms of antibody neutralization ................................................... 23 1.7 Biophysical and biochemical properties of parvovirus capsids ............................. 24 vi 1.8 Critical structures in parvovirus capsids ................................................................ 25 1.9 Endogenous viral elements .................................................................................... 27 1.9a Origin of endogenous viral elements ....................................................... 27 1.9b Identification and dating of endogenous viral elements .......................... 28 1.9c Reconstruction of endogenous viral elements.......................................... 29 1.9d Host suppression of endogenous viral elements ...................................... 30 1.9e Host benefits of endogenous viral elements............................................. 31 1.10 Dissertation overview .......................................................................................... 34 1.11 References ............................................................................................................ 35 CHAPTER TWO: Parvovirus capsid structures required for infection: mutations controlling receptor recognition and protease cleavages ............................................................................ 52 2.1 Abstract .................................................................................................................. 53 2.2 Importance ............................................................................................................. 53 2.3 Introduction ............................................................................................................ 54 2.4 Materials and Methods ........................................................................................... 58 2.5 Results ...................................................................................................................