Structural characterization of spumavirus capsid assemblies

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.