MORPHOLOGICAL ADAPTATIONS FACILITATING ATTACHMENT FOR ARCHAEAL VIRUSES by Ross Alan Hartman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana November 2019 ©COPYRIGHT by Ross Alan Hartman 2019 All Rights Reserved ii DEDICATION This work is dedicated to my daughters Roslyn and Mariah; hope is a little child yet it carries everything. iii ACKNOWLEDGEMENTS I thank all the members of my committee: Mark Young, Martin Lawrence, Valerie Copie, Brian Bothner, and John Peters. I especially thank my advisor Mark for his child-like fascination with the natural world and gentle but persistent effort to understand it. Thank you to all the members of the Young lab past and present especially: Becky Hochstein, Jennifer Wirth, Jamie Snyder, Pilar Manrique, Sue Brumfield, Jonathan Sholey, Peter Wilson, and Lieuwe Biewenga. I especially thank Jonathan and Peter for their hard work and dedication despite consistent and often inexplicable failure. I thank my family for helping me through the darkness by reminding me that sanctification is through suffering, that honor is worthless, and that humility is a hard won virtue. iv TABLE OF CONTENTS 1. INTRODUCTION AND RESEARCH OBJECTIVES ................................................ 1 Archaea The 1st, 2nd, and 3rd Domains of Life ............................................................. 1 Origin and Evolution of Viruses .................................................................................. 4 Influence of Hot Spring Environment on Viral Evolution .......................................... 6 Attachment and Entry for Bacteriophages and Eukaryotic Viruses ............................ 8 Current Knowledge of Attachment and Entry for Archaeal Viruses ......................... 11 Structural Features of Archaeal Viruses and their Functional Roles ......................... 13 Turriviridae and Sphaerolipoviridae ...................................................................18 Rudiviridae and Lipothrixviridae .......................................................................19 Claviviridae .........................................................................................................22 Caudovirales Archaeal Viruses ...........................................................................24 Spindle Viruses: Bicaudaviridae and Fuselloviridae ..........................................24 Biophysical Adaptation to Extreme Environments ............................................27 Research Objectives ................................................................................................... 30 Research Questions .............................................................................................31 2. THE MOLECULAR MECHANISM OF CELLULAR ATTACHMENT FOR AN ARCHAEAL VIRUS .................................................................................... 33 Introduction ................................................................................................................ 33 Materials and Methods .............................................................................................. 36 Quantification of Viral Attachment ....................................................................36 Phylogenetic Tree and Identification of Conserved C381 Residues ..................36 Virus Binding Assay and Imaging ......................................................................37 Cryo-Electron Tomography ................................................................................37 Subvolume Alignment and Averaging for STIV ................................................38 Subvolume Alignment and Averaging for Pili ...................................................39 Subvolume Alignment and Averaging of the Turret/Pilus Interaction ...............39 Fitting the Atomic Structure of STIV into the Subvolume Average ..................40 Isolation of a New STIV-subtype Virus-Host System .......................................40 Generation of STIV Resistant Host Mutants .....................................................42 Sequencing Resistant Mutants and Identification of Mutation Sites ..................42 Results........................................................................................................................ 43 The C381 Crystal Structure ................................................................................43 Structural Similarity ............................................................................................46 Conserved Residues ............................................................................................48 The C381 Subunit Interface ................................................................................51 The A223 Interface .............................................................................................52 The Petal Interface ..............................................................................................52 Virion Adsorption Kinetics .................................................................................53 Initial STIV Attachment to Pili ...........................................................................55 v TABLE OF CONTENTS CONTINUED Identification of Receptor Candidates by Virus Resistant Mutants ...................61 Discussion .................................................................................................................. 66 C557 Petal Protein as a Maturation Factor .........................................................66 An S-layer Glycan Barrier ..................................................................................66 Pilus Identity .......................................................................................................67 Host Discrimination ............................................................................................68 Genome Delivery ................................................................................................68 3. DISCOVERY AND CHARACTERIZATION OF TSPV1: A PILATED ARCHAEAL VIRUS ................................................................................................... 73 Introduction ................................................................................................................ 73 Materials and Methods .............................................................................................. 75 Host and Virus Source ........................................................................................75 Culturing Conditions ..........................................................................................75 Virus Isolation and Purification of Virion Fibers ...............................................76 Cryo-Electron Microscopy .................................................................................77 Genome Sequencing and Assembly ...................................................................78 Gene Prediction and Annotation .........................................................................78 N-terminal Sequencing and LC-MS Sequencing ...............................................79 Capsid Protein Glycosylation Analysis ..............................................................80 Diethyl Ether Sensitivity Assay ..........................................................................81 Results........................................................................................................................ 81 Morphology of TSPV1 .......................................................................................81 TSPV1 Genome Assembly and Annotation .......................................................85 Identification of Major Coat Protein ...................................................................91 Purification of Viral Fibers .................................................................................94 Characterization of the Viral Fibers ...................................................................96 Glycosylation Status of Capsid Proteins .............................................................99 Discussion ................................................................................................................ 100 4. CONCLUSIONS AND FUTURE DIRECTIONS ................................................... 105 STIV Research Conclusions and Future Directions ............................................... 106 Final Remarks and Future Directions for TSPV1 .................................................... 107 APPENDICIES ............................................................................................................... 126 APPENDIX A: Supplemental Tables and Figures from . Chapter Two ................................................................................... 127 APPENDIX B: Custom Scripts for Sub-Tomogram Averaging ............................. 137 vi TABLE OF CONTENTS CONTINUED APPENDIX C: Custom Scripts for Movie Generation . in Chimera ..................................................................................... 142 vii LIST OF TABLES Table Page 1.1. Archaeal Viruses with Structural Data for Their Capsids . and Coat Proteins ............................................................................................... 16 1.2. Archaeal Virus Families and Their Adaptations to . Extreme Environment .....................................................................................