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Item Morphological adaptations facilitating attachment for archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2019) Hartman, Ross Alan; Chairperson, Graduate Committee: Mark J. YoungLittle is known regarding the attachment and entry process for any archaeal virus. The virus capsid serves multiple biological functions including: to protect the viral genome during transit between host cells, and to facilitate attachment and entry of the viral genome to a new host cell. Virus attachment is conducted without expenditure of stored chemical energy i.e. ATP hydrolysis. Instead, virus particles depend on diffusion for transportation and attachment from one host cell to another. This thesis examines the attachment process for two archaeal viruses. Sulfolobus turreted icosahedral virus (STIV) is well characterized for an archaeal virus. Still, no information is available concerning STIV attachment or entry. The research presented here shows that STIV attaches to a host cell pilus. Furthermore, combining the previously determined atomic model for the virus, with cryo-electron tomography, a pseudo-atomic model of the interaction between the host pilus and virus was determined. Based on this data, a model is proposed for the maturation of the virus capsid from a noninfectious to an infectious form, by dissociation of accessory proteins. Finally, a locus of genes is identified in the host cell, encoding proteins essential for viral infection, that are likely components of the pili structure recognized by STIV. The isolation of a new archaeal virus, Thermoproteus Piliferous Virus 1 (TSPV1), is also presented here. The TSPV1 virion has numerous fibrous extensions from the capsid, of varying length, that are the first observed for any virus. The capsid 2-3nm fibers likely serve to extend the effective surface area of the virus, facilitating attachment to host cells. Characterization of this new virus was conducted, including genome sequencing and determination of the protein identity for the capsid fibers. The research presented here provides a substantial advancement in our knowledge of the attachment process for archaeal viruses. Attachment to host pili is now emerging as a common theme for archaeal viruses. Furthermore, the isolation of the new archaeal virus TSPV1 demonstrates a novel strategy to increase the probability of interaction between a virus and host cell.Item Virus host interactions at the single cell level in hot springs of Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2019) Munson-McGee, Jacob Hampton; Chairperson, Graduate Committee: Mark J. Young; Jamie C. Snyder and Mark J. Young were co-authors of the article, 'Introduction to archaeal viruses' in the journal 'Genes' which is contained within this dissertation.; Ross Hartman was an author and Mark J. Young were co-authors of the article, 'vFish for the quantification of viral infection in natural environments' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Erin K. Field, Mary Bateson, Colleen Rooney, Ramunas Stepanauskas and Mark J. Young were co-authors of the article, 'The identification and characterization of a nanoarchaeota, its cellular host and a nanoarchaeal virus across Yellowstone National Park hot springs' which is contained within this dissertation.; Colleen Rooney and Mark J. Young were co-authors of the article, 'An uncultivated virus infecting a nanoarchaeal parasite in the hot springs of Yellowstone National Park' submitted to the journal 'Virology' which is contained within this dissertation.; Shengyun Peng, Samantha Dewerff, Ramunas Stepanauskas, Rachel J. Whitaker, Joshua Weitz and Mark J. Young were co-authors of the article, 'A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments' in the journal 'The ISME journal' which is contained within this dissertation.Viruses are the most abundant biological entities on the planet and virus-host interactions are some of the most important factors in shaping microbial community structure and function and global chemical cycling. The high temperature low pH hot spring of Yellowstone National Park contain simplified microbial communities of 8-10 Archaeal species, and comparatively simple viral communities. These idealized communities that contain only viruses and their Archaeal hosts represent a model natural environment for the study of viruses and their hosts. This work presented here builds on previous population level studies of the viral and microbial communities to examine virus-host interactions at the single cell level. The identification of viral infection has long been a scourge of environmental virologist. In order to identify viral infection in natural environments we have adapted Fluorescent in situ hybridization (FISH) techniques to directly identify viral sequences. We further advance this technique to be compatible with flow cytometry analysis for the rapid quantification of viral infection of uncharacterized viruses in natural environments. This technique is used to quantify viral infection of two different viruses, previously only characterized by metagenomic sequencing analysis, in four geographically separate low pH high temperature hot springs of Yellowstone National Park. Finally, we combine viral and cellular metagenomics with cellular transcriptomics and single cell genomics to identify virus host interactions at the single cell level and identify viruses that are replicating in the hot springs. This work suggests that a majority of cells in the hot springs are interacting with viruses and that a majority of the cells are interacting with multiple viruses at any given time. We also identify RNA sequences from a majority of the viral types present in the hot springs suggesting that viral replication is occurring and is an important force in determining the structure and function of the microbial communities in these hot springs. Together these works represent a significant advancement of our understanding of virus host interactions in natural environments as well as new techniques to be used in future studies.Item Insect microbe interactions: honey bee antiviral defense mechanisms and characterization of Spiroplasma colonizing wheat stem sawfly(Montana State University - Bozeman, College of Agriculture, 2017) Brutscher, Laura Marie; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Virus and DSRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense' in the journal 'Scientific reports' which is contained within this thesis.; Curtis Fowler, David K. Weaver and Carl J. Yeoman were co-authors of the article, 'Identification and characterization of a Spiroplasma sp. (Ixodetis clade) associated with the wheat stem sawfly (Cephus cinctus)' submitted to the journal 'Microbial ecology' which is contained within this thesis.Insects play important roles in ecosystems throughout the world. There are many beneficial insects, including those that pollinate plants in diverse landscapes, while other insects are considered agricultural pests. Regardless of ecological role, insects are hosts for microbial symbionts and pathogens. Some microorganisms (e.g., viruses) are harmful to insect health, but many microbial symbionts aid in host biological processes. The projects herein describe the interplay between insects and microbes; specifically (1) honey bee host - virus interactions and (2) identification and characterization of a wheat stem sawfly-associated Spiroplasma. Honey bees (Apis mellifera) are pollinators of numerous agricultural crops and other plant species. Since 2006, there have been high annual losses of honey bee colonies in the U.S. (~33%) and throughout the world. Colony deaths are influenced by multiple factors including RNA virus infections. Honey bee antiviral defense involves several immune pathways, including dsRNA mediated responses, (i.e., RNA interference and non-sequence-specific dsRNA-triggered responses), but their relative importance in antiviral defense is not well understood. To investigate honey bee antiviral defense, bees were infected with model virus in the absence or presence of dsRNA, which reduced virus abundance. Transcriptome-level analysis determined hundreds of genes were differentially expressed in response to co-treatment of dsRNA and virus, including immune-related genes. RNAi-mediated gene knockdown of two putative antiviral genes increased virus abundance and supported their antiviral role. Additional investigation of these and other genes will improve our understanding of dsRNA-mediated antiviral defense in honey bees. In contrast, wheat stem sawflies (Cephus cintus) are major wheat pests in the Northwest United States. Strategies that target endosymbionts of sawflies could reduce wheat crop losses. Hereunto, the microbes that colonize wheat stem sawfly have not been explored. Targeted DNA sequencing determined sawflies were colonized by a Spiroplasma species that has greatest 16S rRNA sequence identity with Ixodetis clade species. Metagenomic sequencing identified several Spiroplasma encoded genes involved in metabolism, which may be important to the sawfly host. Further characterization of honey bee-virus interactions and the role of Spiroplasma in sawfly health may contribute to limiting threats to global crop production and will further scientific understanding of non-model insect-microbe interactions.Item The intercellular spread of alphaherpesviruses(Montana State University - Bozeman, College of Letters & Science, 2018) Herr, Alix Elise; Chairperson, Graduate Committee: Matt Taylor; Kyles S. Hain and Matthew P. Taylor were co-authors of the article, 'Limitations on the multiplicity of cellular infection during human alphaherpesviral disease' in the journal 'Current clinical microbiology reports' which is contained within this thesis.; Theresa Thornburg, Max DePartee, Ryan Waters and Matthew P. Taylor were co-authors of the article, 'The route of transmission from neurons impacts pseudorabies virus coinfection in vivo' submitted to the journal 'Journal of virology' which is contained within this thesis.There are three prominent alphaherpesviruses that infect humans: Herpes Simplex virus-1, Herpes Simplex virus-2, and Varicella Zoster virus. Each virus is harbored within 15-98% of the population. Prototypical infections involve only a handful of intercellular spread events within a host. Most spread events involve neurons. Only one virion is thought to be successfully transmitted from a neuron to another cell -- but this has yet to be verified during infectious spread within a host. In this dissertation, we used Pseudorabies virus to trace the number of virions spreading infection from infected neurons to uninfected cells. Pseudorabies virus is a prominent model alphaherpesvirus that infects mice. To quantify the number of virions transmitted between cells, we intravitreally injected different, genetically engineered Pseudorabies virus strains and quantified spread to the murine central nervous system. We calculated the average number of expressed viral genomes per infected neuron by utilizing the Poisson distributions of neurons expressing one, two, or three different viral strains. We found that when a neuronal axon transmitted infection to cells, a cell became infected with one virion on average. In contrast, when neuronal soma or dendrites transmitted the viral strains to surrounding cells, each cell expressed three viral genomes on average. Most importantly, we discovered that the absence of a specific alphaherpesviral protein, US9, diminished the capacity of the virus to infect a cell with a plurality of viral particles. This dissertation advanced the field of herpesviral research in two ways: by quantifying the number of alphaherpesviral particles transmitted between infected neurons in a host and identifying a viral protein instrumental in determining the number virions transmitted from neurons to other cells. The average number of viral particles infecting cells within a host determines the viral genetic dosage and impacts viral gene expression, viral replicative rates, and viral diversification.Item Biophysical characterization of P22 bacteriophage and adenoassociated viruses(Montana State University - Bozeman, College of Letters & Science, 2016) Kant, Ravi; Chairperson, Graduate Committee: Brian Bothner; Aida Llauro, Vamseedhar Rayaprolu, Shefah Qazi, Pedro J. de Pablo, Trevor Douglas and Brian Bothner were co-authors of the article, 'Stability, biomechanics and structural changes in P22 bacteriophage during maturation' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Understanding of P22 bacteriophage maturation by QCM-D' which is contained within this thesis.; Navid Movahed, Dewey Brooke, Antonette Bennett, Mavis Agbandje-McKenna and Brian Bothner were co-authors of the article, 'Prolonged incubation with liposome leads to PLA2 activation in adeno-associated viruses' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Comparison of the visco-elastic properties of viruses, virus based nanomaterials and active protein cages' which is contained within this thesis.The dsDNA tailed bacteriophages comprise the largest evolving life form in the biosphere. They are not only the most abundant organism on Earth, but also plausibly the most ancient. The ancient origin of phage suggests that they have had the ample opportunity to undergo the evolutionary changes necessary to perform intricate coordinated biological functions. Therefore, characterizing a tailed bacteriophage will help not only to understand biology, but also help us to establish a relationship between structure and function. Viruses display a dynamic equilibrium between structural conformations, stability, flexibility and rigidity which is essential for the perpetuation of life cycle. Understanding this complex biophysical relationship is a daunting task and requires a combination of multidimensional approaches. P22 is a tailed bacteriophage and displays a series of structural transitions during maturation. To understand the important biophysical changes in the P22 at different stages of maturation, we have introduced a suite of orthogonal techniques to address the distinct properties of intermediates. These include Differential Scanning Fluorimetry which probes the thermal stability of P22 capsids, Hydrogen-Deuterium Mass Spectrometry, which probes the conformational flexibility and Atomic Force Microscopy and Quartz Crystal Microbalance with dissipation, which probe the biomechanical transformation in the capsids. P22 investigation using these techniques reveals the large scale structural arrangements along with the expansion. Global rearrangement results in an increase in stability, rigidity and reduced dynamics. The sum results of these studies indicate that expansion is accompanied by large scale inter-subunit rearrangements which lead to the enhanced hydrophobic core at different quasi-equivalent axes. We have also studied Adeno-associated viruses, which is used as a gene delivery vehicle for the treatment of genetic disorders. AAVs lipase contains a lipase domain and its activation is important for the successful infection. Activation mechanism of lipase domain is not thoroughly understood. To understand the mechanism, we have developed a Liquid Chromatography-Mass Spectrometry assay sensitive enough to measure lipase products. This assay confirms that prolonged incubation of AAVs with liposome is able to activate the lipase domain without the involvement of receptors and co-receptors.Item Biological redesign of virus particles for a new era of catalytic materials(Montana State University - Bozeman, College of Letters & Science, 2016) Jordan, Paul Campion; Chairperson, Graduate Committee: Trevor Douglas; Dustin P. Patterson, Kendall N. Saboda, Ethan J. Edwards, Heini M. Miettinen, Gautam Basu, Megan C. Thieleges and Trevor Douglas were co-authors of the article, 'Self-assembling biomolecular catalysts for hydrogen production' in the journal 'Nature chemistry' which is contained within this dissertation.; Joseph C-Y Wang, Ethan J. Edwards, Heini M. Miettinen, Amanda L. Le Sueur, Megan C. Thielges , Adam Zlotnick and Trevor Douglas were co-authors of the article, 'Redesign of a virus particle for NADH-driven hydrogen production' which is contained within this dissertation.; This dissertation contains one article of which Paul Campion Jordan is not the main author.Biology has designed a suite of compartments and barriers that confine fundamental biochemical reactions. Such barriers include the membrane-bound organelles but also a suite of protein-based compartments that architecturally and chemically integrate catalytic processes. These compartments co-polymerize from multiple protein subunits to form polyhedral structures that spatially separate enzymatic processes. Protein compartments confine volatile intermediates, trap toxic reaction products, and co-localize multiple enzymatic processes for catalytic enhancements. The protein-based compartments represent, advantageously, a combination of form and function that has inspired the synthesis of new, designer materials. The self-assembly of cage-like structures, the structures of which are reminiscent of the compartments, has been used for the directed encapsulation of active enzymes. We have used the capsid from bacteriophage P22, as a nanocontainer for directing the encapsulation of a variety of gene products, including active enzymes. The P22 capsid assembles from a coat protein (CP) and a scaffold protein (SP) which templates its assembly. Using the simplicity of the P22 expression system, a strategy was developed and implemented for the directed encapsulation of an active, [NiFe] hydrogenase. We hypothesized and proved the enzyme active site needed to be matured by accessory proteins found within the expression host. A two plasmid expression system was designed, where the hydrogenase cargo was under the control of a different inducer than the P22 CP. The [NiFe]-hydrogenase is a heterodimer and each enzyme subunit was fused to different SP. The resultant packaging of the two SP fusions, with the hydrogenase large and small subunits fused to them stabilized a weak heterodimeric structure. Remarkably, the stabilizing effects of the capsid allowed us to probe the infrared signatures associated with the hydrogenase active site. Finally, the progress made here in developing a virus capsid for H2 production left room to build increased complexity into the P22-Hydrogenase system while also taking inspiration from the innate, biological function of the hydrogenase. We incorporated a cytochrome/cytochrome reductase pair to drive H 2 production using NADH. These designs, built at the molecular level, represent inherently renewable catalysts that pave the way for a new era of catalytic materials synthesized entirely by biology.Item The discovery and characterization of two archaeal viruses using culture-independent methods(Montana State University - Bozeman, College of Letters & Science, 2015) Hochstein, Rebecca Ann; Chairperson, Graduate Committee: Mark J. Young; Daniel Bollschweiller, Harald Engelhardt, C. Martin Lawrence, and Mark Young were co-authors of the article, 'Large tailed spindle viruses of archaea: a new way of doing viral business' in the journal 'Journal of virology' which is contained within this thesis.The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content, but rarely provide knowledge about virion morphology and or cellular host identity. This thesis describes research to utilize culture-independent methods to identify and to characterize two new archaeal viruses starting with viral metagenomic sequences. The first virus, Acidianus tailed spindle virus (ATSV), was initially identified by bioinformatic analysis of viral metagenomic datasets from a high temperature (80° C) acidic (pH 2) hot spring located in Yellowstone National Park, USA. ATSV was purified and characterized directly from environmental samples without dependency on culturing. Characterization included identification of the large tailed spindle shape virion morphology, determination of the complete 70.8 kbp circular ds DNA viral genome content, and identification of its cellular host. The host of ATSV, Acidianus hospitalis, was determined using CRISPR/Cas identification and CARD-FISH, and was confirmed by culturing. Additional characterization of ATSV included solving the structure of the major coat protein (MCP) by X-ray crystallography. The ATSV MCP reveals a decorated right-handed four helix bundle. The MCP is packed into the crystal as a four-start superhelix, for which the interfaces show biologically relevant interactions, indicating that ATSV might assemble using a multi-start helix. CryoEM images of ATSV show striations extending across the virion, supporting an assembly model in which long protein strands form the spindle virion structure. This is the first known model of spindle virus assembly. Culture-independent methods developed for ATSV purification and characterization were applied to a second virus, a pleomorphic particle found in high abundance in the CHAS viral fraction. Using mass spectrometry identification, viral metagenomics, deep sequencing, and host identification, a full virus genome and a host were linked to the virus particle, named Stygiolobus pleomorphic virus (SPV). SPV most likely represents a new virus family, with a unique particle morphology and gene content. Taken together, the results reported in this thesis provide an expanded pathway for the discovery, isolation and characterization of new viruses using culture-independent approaches.Item Partial characterization of an entomopoxvirus isolated from grasshoppers(Montana State University - Bozeman, College of Agriculture, 1978) Kussman, Herbert CarlItem Homology of the spheroidin gene from entomopoxviruses isolated from Melanoplus sanguinipes and Amsacta moorei(Montana State University - Bozeman, College of Agriculture, 1994) Wilson, SuzanneItem The effects of the bovine respiratory syncytial virus on the ciliated epithelium of fetal bovine tracheal organ culture(Montana State University - Bozeman, College of Agriculture, 1979) Cantrell, Charles Garrett