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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 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 Understanding the solution-phase biophysics and conformational dynamics of virus capsids(Montana State University - Bozeman, College of Letters & Science, 2013) Rayaprolu, Vamseedhar; Chairperson, Graduate Committee: Brian Bothner; Benjamin M. Manning, Trevor Douglas and Brian Bothner were co-authors of the article, 'Virus particles as active nanomaterials that can rapidly change their viscoelastic properties in response to dilute solutions' in the journal 'RSC softmatter' which is contained within this thesis.; Shannon Kruse, Navid Movahed, Tim Potter, Balasubramanian Venkatakrishnan, Bridget Lins, Antonette Bennett, Robert McKenna, Mavis Agbandje-McKenna and Brian Bothner were co-authors of the article, 'Comparative analysis of adeno associated virus capsid stability and dynamics' submitted to the journal 'Journal of virology' which is contained within this thesis.; Navid Movahed, Ravikant Chaudhary, Geoff Blatter, Alec Skuntz, Sue Brumfield, Jonathan K. Hilmer, Mark J. Young, Trevor Douglas and Brian Bothner were co-authors of the article, 'Learning new tricks from an old dog; studies of CCMV capsid swelling' submitted to the journal 'Journal of virology' which is contained within this thesis.Viruses are the most abundant form of life on the planet. Many forms are pathogenic and represent a major threat to human health, but viruses recently have been used as nanoscale tools for gene therapy, drug delivery and enzyme nanoreactors. Viruses have historically been viewed as static and rigid delivery vehicles, but over the last few decades they have been recognized as flexible structures. Their structural dynamics are a crucial element of their functionality. Characterizing the biophysical properties of these viruses is both challenging and exciting. We have developed and used a multidimensional approach to tackle this task. Our techniques include Differential Scanning Fluorimetry, which probes the melting temperatures of virus capsids by the use of a fluorescent dye and Hydrogen-Deuterium Mass Spectrometry, which investigates the flexibility of the virus capsid protein by following the change in mass when Hydrogen is exchanged with Deuterium. Flexible regions exchange more. The above techniques are well complemented by the use of size-exclusion chromatography, which differentiates virus capsids based on their hydrodynamic radius and Limited proteolysis which again probes dynamic regions of the capsids up to the amino acid level. We have studied two different systems, Cowpea chlorotic mottle virus (CCMV) and Adeno-associated virus (AAV) using these methodologies. The sum result of these assays indicate that, in case of CCMV, capsids can undergo structural transitions due to very subtle pH and cation concentrations and the capsid protein is capable of rigid body transitions which affect the stability, while maintaining most of the secondary structure. In the case of AAV, the inherent sequence differences explains only partially the differences in stability and proteolytic susceptibility.