{"acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"ScienComp"}],"day":"26","title":"Structural characterization of spumavirus capsid assemblies","supervisor":[{"orcid":"0000-0003-4790-8078","full_name":"Schur, Florian KM","id":"48AD8942-F248-11E8-B48F-1D18A9856A87","first_name":"Florian KM","last_name":"Schur"}],"abstract":[{"lang":"eng","text":"The Retroviridae family consists of two sub-families, the Orthoretrovirinae and the\r\nSpumaretrovirinae. The Orthoretroviruses contain important human pathogens, such as the\r\nhuman immunodeficiency virus 1 (HIV-1). They also harbor other retrovirus species which\r\nare regularly used as model systems to study the retroviral life cycle. The main structural\r\ncomponent of the retroviruses, is the Gag protein and its truncation derivatives occurring\r\nduring viral maturation. Orthoretroviral Gag assemblies have been extensively studied to\r\nunderstand the interactions that confer stability and morphology to viral particles.\r\nThe Spumaretrovirinae subfamily represent an early diverging branch of the Retroviridae.\r\nIts members, the Foamy viruses (FV), share most of the conventional features found in\r\nretroviruses. However, they also possess multiple characteristics that make them unique. In\r\nparticular, FV Gag does not get extensively cleaved as in orthoretroviruses. Hence, the Gag\r\narchitecture deviates from the canonical domain arrangement in FV. They also exhibit a\r\npeculiar particle morphology, having no apparent immature state and a seemingly\r\nicosahedral mature particle. Due to this, many fundamental questions on FV structural\r\nassembly mechanisms remain open. To answer these questions, was the main focus of this\r\nthesis.\r\nMainly, it is not known how FV assemble their core in a virus particle and what are the\r\nimportant assembly interaction sites within said core. What is the minimum assembly\r\ncompetent 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\r\ninteresting to know what is the evolutionary relationship between FV and the rest of the\r\nretrotranscribing elements, from a structural point of view?\r\nTo answer these questions, membrane-enveloped mammalian cell-derived FV virus-like\r\nparticles (VLPs) were produced. Cryo-electron tomography (cryo-ET) analysis suggested\r\nthese FV VLPs do not form a canonical retroviral Gag lattice structure, which is in line with\r\nearlier observations. To further evaluate FV Gag assembly competence and morphology,\r\nthe first bacterial cell-derived in vitro VLP assembly system was designed and optimized.\r\nUsing this system with different truncation variants, the minimum assembly competent\r\ndomain of FV Gag was found to be the putative CA300-477 domain. Varying VLP\r\nmorphologies were also observed and strongly suggested residues upstream of CA300-477\r\nplay 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\r\nassembly competent domain. This revealed an unexpectedly unique non-canonical\r\nassembly architecture. Three novel lattice stabilizing interfaces were described which\r\nproved to be as unique as the lattice arrangement. Comparison to a newly published FV CA\r\ncore structure revealed the CA-CA interactions in the atypical assembly do not recapitulate\r\nwhat is described for the FV core lattice. However, the new in vitro VLP assembly system\r\nobtained in this thesis also provides an exciting opportunity to study still unresolved FV\r\nassembly features in a potentially facilitated approach compared to conventional methods.\r\nIn summary, this work provided a deeper understanding of the basic FV Gag assembly unit,\r\nas well as presenting the first FV Gag-derived in vitro VLP assembly system. This system\r\nreveals a novel and unique assembly architecture among retroviral in vitro assemblies."}],"date_created":"2024-09-20T10:21:03Z","date_published":"2024-09-26T00:00:00Z","doi":"10.15479/at:ista:18101","oa_version":"Published Version","project":[{"grant_number":"665385","call_identifier":"H2020","name":"International IST Doctoral Program","_id":"2564DBCA-B435-11E9-9278-68D0E5697425"},{"name":"Structural characterization of spumavirus capsid assemblies to understand conserved Ortervirales assembly mechanisms","_id":"9B9C98E0-BA93-11EA-9121-9846C619BF3A","grant_number":"25762"}],"article_processing_charge":"No","alternative_title":["ISTA Thesis"],"ddc":["570"],"page":"131","language":[{"iso":"eng"}],"file":[{"access_level":"closed","date_created":"2024-09-26T13:40:33Z","relation":"source_file","file_size":14213128,"checksum":"3b8b0bacfe61112f3852744f3170e468","embargo_to":"open_access","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_name":"PhD_thesis_DPorley_final_20240919.docx","creator":"dporley","date_updated":"2025-03-25T23:30:03Z","file_id":"18149"},{"file_size":18583031,"relation":"main_file","access_level":"open_access","date_created":"2024-09-26T13:41:39Z","checksum":"6c3a652a8eede874118e11d66a63652f","content_type":"application/pdf","embargo":"2025-03-25","file_name":"PhD_thesis_DPorley_final_20240926_pdfa1.pdf","creator":"dporley","file_id":"18150","date_updated":"2025-03-25T23:30:03Z"}],"year":"2024","oa":1,"status":"public","citation":{"chicago":"Porley Esteves, Darío. “Structural Characterization of Spumavirus Capsid Assemblies.” Institute of Science and Technology Austria, 2024. <a href=\"https://doi.org/10.15479/at:ista:18101\">https://doi.org/10.15479/at:ista:18101</a>.","ieee":"D. Porley Esteves, “Structural characterization of spumavirus capsid assemblies,” Institute of Science and Technology Austria, 2024.","ama":"Porley Esteves D. Structural characterization of spumavirus capsid assemblies. 2024. doi:<a href=\"https://doi.org/10.15479/at:ista:18101\">10.15479/at:ista:18101</a>","short":"D. Porley Esteves, Structural Characterization of Spumavirus Capsid Assemblies, Institute of Science and Technology Austria, 2024.","apa":"Porley Esteves, D. (2024). <i>Structural characterization of spumavirus capsid assemblies</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:18101\">https://doi.org/10.15479/at:ista:18101</a>","ista":"Porley Esteves D. 2024. Structural characterization of spumavirus capsid assemblies. Institute of Science and Technology Austria.","mla":"Porley Esteves, Darío. <i>Structural Characterization of Spumavirus Capsid Assemblies</i>. Institute of Science and Technology Austria, 2024, doi:<a href=\"https://doi.org/10.15479/at:ista:18101\">10.15479/at:ista:18101</a>."},"corr_author":"1","publication_identifier":{"isbn":["978-3-99078-041-1"],"issn":["2663-337X"]},"ec_funded":1,"author":[{"id":"2FD6EA6C-F248-11E8-B48F-1D18A9856A87","last_name":"Porley","first_name":"Dario J","full_name":"Porley, Dario J"}],"file_date_updated":"2025-03-25T23:30:03Z","has_accepted_license":"1","publisher":"Institute of Science and Technology Austria","_id":"18101","department":[{"_id":"GradSch"},{"_id":"FlSc"}],"month":"09","date_updated":"2025-05-14T11:26:43Z","type":"dissertation","publication_status":"published","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","degree_awarded":"PhD"}