000			03840ntm a22003737a 4500
003			AT-ISTA
005			20250915132911.0
008			250915s2024 au ||||| m||| 00| 0 eng d
040			_cISTA
100			_aDatler, Julia _91084230
245			_aElucidating the structural determinants of the poxvirus core using multi-modal cryo-EM
260			_bInstitute of Science and Technology Austria _c2024
500			_aThesis
505			_aAbstract
505			_aAcknowledgements
505			_aAbout the Author
505			_aList of Publications
505			_aList of Contributors
505			_aTable of Contents
505			_aList of Figures
505			_aList of Tables
505			_aList of Abbreviations
505			_a1 Introduction
505			_a2 Aims and Objectives
505			_a3 Material and Methods
505			_a4 Results
505			_a5 Discussion
505			_aReferences
505			_aA Appendix 1
505			_aB Appendix 2
520			_aPoxviruses 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.
856			_uhttps://doi.org/10.15479/at:ista:18766
942			_2ddc
999			_c768069 _d768069