@phdthesis{18766, abstract = {Poxviruses are large pleomorphic double-stranded DNA viruses that include well known members such as variola virus, the causative agent of smallpox, Mpox virus, as well as Vaccinia virus (VACV), which serves as a vaccination strain for formerly mentioned viruses. VACV is a valuable model for studying large pleomorphic DNA viruses in general and poxviruses specifically, as many features, such as core morphology and structural proteins, are well conserved within this family. Despite decades of research, our understanding of the structural components and proteins that comprise the poxvirus core in mature virions remains limited. Although major core proteins were identified via indirect experimental evidence, the core's complexity, with its large size, structure and number of involved proteins, has hindered efforts to achieve high-resolution insights and to define the roles of the individual proteins. The specific protein composition of the core's individual layers, including the palisade layer and the inner core wall, has remained unclear. In this study, we have merged multiple approaches, including single particle cryo electron microscopy of purified virus cores, cryo-electron tomography and subtomogram averaging of mature virions and molecular modeling to elucidate the structural determinants of the VACV core. Due to the lack of experimentally derived structures, either in situ or reconstituted in vitro, we used Alphafold to predict models of the putative major core protein candidates, A10, 23k, A3, A4, and L4. Our results show that the VACV core is composed of several layers with varying local symmetries, forming more intricate interactions than observed previously. This allowed us to identify several molecular building blocks forming the viral core lattice. In particular, we identified trimers of protein A10 as a major core structure that forms the palisade layer of the viral core. Additionally, we revealed that six petals of a flower shaped core pore within the core wall are composed of A10 trimers. Furthermore, we obtained a cryo-EM density for the inner core wall that could potentially accommodate an A3 dimer. Integrating descriptions of protein interactions from previous studies enabled us to provide a detailed structural model of the poxvirus core wall, and our findings indicate that the interactions within A10 trimers are likely consistent across orthopox- and parapoxviruses. This combined application of cryo-SPA and cryo-ET can help overcome obstacles in studying complex virus structures in the future, including their key assembly proteins, interactions, and the formation into a core lattice. Our work provides important fundamental new insights into poxvirus core architecture, also considering the recent re-emergence of poxviruses.}, author = {Datler, Julia}, isbn = {978-3-99078-049-7}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, cryo-SPA, Structural Virology, Poxvirus, Vaccinia Virus, Structural Biology}, pages = {106}, publisher = {Institute of Science and Technology Austria}, title = {{Elucidating the structural determinants of the poxvirus core using multi-modal cryo-EM}}, doi = {10.15479/at:ista:18766}, year = {2024}, } @phdthesis{18101, abstract = {The Retroviridae family consists of two sub-families, the Orthoretrovirinae and the Spumaretrovirinae. The Orthoretroviruses contain important human pathogens, such as the human immunodeficiency virus 1 (HIV-1). They also harbor other retrovirus species which are regularly used as model systems to study the retroviral life cycle. The main structural component of the retroviruses, is the Gag protein and its truncation derivatives occurring during viral maturation. Orthoretroviral Gag assemblies have been extensively studied to understand the interactions that confer stability and morphology to viral particles. The Spumaretrovirinae subfamily represent an early diverging branch of the Retroviridae. Its members, the Foamy viruses (FV), share most of the conventional features found in retroviruses. However, they also possess multiple characteristics that make them unique. In particular, FV Gag does not get extensively cleaved as in orthoretroviruses. Hence, the Gag architecture deviates from the canonical domain arrangement in FV. They also exhibit a peculiar particle morphology, having no apparent immature state and a seemingly icosahedral mature particle. Due to this, many fundamental questions on FV structural assembly mechanisms remain open. To answer these questions, was the main focus of this thesis. Mainly, it is not known how FV assemble their core in a virus particle and what are the important assembly interaction sites within said core. What is the minimum assembly competent domain of FV Gag? Is there a morphological change in the assembly type of FVGag lattices? If so, what is defining these morphological shifts? Finally, it would be interesting to know what is the evolutionary relationship between FV and the rest of the retrotranscribing elements, from a structural point of view? To answer these questions, membrane-enveloped mammalian cell-derived FV virus-like particles (VLPs) were produced. Cryo-electron tomography (cryo-ET) analysis suggested these FV VLPs do not form a canonical retroviral Gag lattice structure, which is in line with earlier observations. To further evaluate FV Gag assembly competence and morphology, the first bacterial cell-derived in vitro VLP assembly system was designed and optimized. Using this system with different truncation variants, the minimum assembly competent domain of FV Gag was found to be the putative CA300-477 domain. Varying VLP morphologies were also observed and strongly suggested residues upstream of CA300-477 play a role in morphology determination. Finally, a combined cryo-electron microscopy (cryoEM) and cryo-ET approach was taken to analyze tubular assemblies from the minimal assembly competent domain. This revealed an unexpectedly unique non-canonical assembly architecture. Three novel lattice stabilizing interfaces were described which proved to be as unique as the lattice arrangement. Comparison to a newly published FV CA core structure revealed the CA-CA interactions in the atypical assembly do not recapitulate what is described for the FV core lattice. However, the new in vitro VLP assembly system obtained in this thesis also provides an exciting opportunity to study still unresolved FV assembly features in a potentially facilitated approach compared to conventional methods. In summary, this work provided a deeper understanding of the basic FV Gag assembly unit, as well as presenting the first FV Gag-derived in vitro VLP assembly system. This system reveals a novel and unique assembly architecture among retroviral in vitro assemblies.}, author = {Porley, Dario J}, isbn = {978-3-99078-041-1}, issn = {2663-337X}, pages = {131}, publisher = {Institute of Science and Technology Austria}, title = {{Structural characterization of spumavirus capsid assemblies}}, doi = {10.15479/at:ista:18101}, year = {2024}, } @phdthesis{12491, abstract = {The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.}, author = {Zens, Bettina}, isbn = {978-3-99078-027-5}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, FIB milling, method development, FIBSEM, extracellular matrix, ECM, cell-derived matrices, CDMs, cell culture, high pressure freezing, HPF, structural biology, tomography, collagen}, pages = {187}, publisher = {Institute of Science and Technology Austria}, title = {{Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography}}, doi = {10.15479/at:ista:12491}, year = {2023}, }