@article{17884, abstract = {Human T cell leukemia virus type 1 (HTLV-1) immature particles differ in morphology from other retroviruses, suggesting a distinct way of assembly. Here we report the results of cryo-electron tomography studies of HTLV-1 virus-like particles assembled in vitro, as well as derived from cells. This work shows that HTLV-1 uses a distinct mechanism of Gag–Gag interactions to form the immature viral lattice. Analysis of high-resolution structural information from immature capsid (CA) tubular arrays reveals that the primary stabilizing component in HTLV-1 is the N-terminal domain of CA. Mutagenesis analysis supports this observation. This distinguishes HTLV-1 from other retroviruses, in which the stabilization is provided primarily by the C-terminal domain of CA. These results provide structural details of the quaternary arrangement of Gag for an immature deltaretrovirus and this helps explain why HTLV-1 particles are morphologically distinct.}, author = {Obr, Martin and Percipalle, Mathias and Chernikova, Darya and Yang, Huixin and Thader, Andreas and Pinke, Gergely and Porley, Dario J and Mansky, Louis M. and Dick, Robert A. and Schur, Florian KM}, issn = {1545-9985}, journal = {Nature Structural & Molecular Biology}, pages = {268--276}, publisher = {Springer Nature}, title = {{Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice}}, doi = {10.1038/s41594-024-01390-8}, volume = {32}, year = {2025}, } @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}, } @article{14979, abstract = {Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.}, author = {Datler, Julia and Hansen, Jesse and Thader, Andreas and Schlögl, Alois and Bauer, Lukas W and Hodirnau, Victor-Valentin and Schur, Florian KM}, issn = {1545-9985}, journal = {Nature Structural & Molecular Biology}, keywords = {Molecular Biology, Structural Biology}, pages = {1114--1123}, publisher = {Springer Nature}, title = {{Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores}}, doi = {10.1038/s41594-023-01201-6}, volume = {31}, year = {2024}, } @article{14255, abstract = {Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.}, author = {Koch, Jana and Xin, Qilin and Obr, Martin and Schäfer, Alicia and Rolfs, Nina and Anagho, Holda A. and Kudulyte, Aiste and Woltereck, Lea and Kummer, Susann and Campos, Joaquin and Uckeley, Zina M. and Bell-Sakyi, Lesley and Kräusslich, Hans Georg and Schur, Florian Km and Acuna, Claudio and Lozach, Pierre Yves}, issn = {1553-7374}, journal = {PLoS Pathogens}, number = {8}, publisher = {Public Library of Science}, title = {{The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells}}, doi = {10.1371/journal.ppat.1011562}, volume = {19}, year = {2023}, } @article{10639, abstract = {With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30-40 min. The virus entered Rab5a+ early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15-25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration.}, author = {Windhaber, Stefan and Xin, Qilin and Uckeley, Zina M. and Koch, Jana and Obr, Martin and Garnier, Céline and Luengo-Guyonnot, Catherine and Duboeuf, Maëva and Schur, Florian KM and Lozach, Pierre-Yves}, issn = {1098-5514}, journal = {Journal of Virology}, keywords = {virology, insect science, immunology, microbiology}, number = {5}, publisher = {American Society for Microbiology}, title = {{The Orthobunyavirus Germiston enters host cells from late endosomes}}, doi = {10.1128/jvi.02146-21}, volume = {96}, year = {2022}, } @article{11155, abstract = {The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.}, author = {Obr, Martin and Hagen, Wim J.H. and Dick, Robert A. and Yu, Lingbo and Kotecha, Abhay and Schur, Florian KM}, issn = {1047-8477}, journal = {Journal of Structural Biology}, keywords = {Structural Biology}, number = {2}, publisher = {Elsevier}, title = {{Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs}}, doi = {10.1016/j.jsb.2022.107852}, volume = {214}, year = {2022}, } @article{10103, abstract = {The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses.}, author = {Obr, Martin and Schur, Florian KM and Dick, Robert A.}, issn = {1999-4915}, journal = {Viruses}, keywords = {virology, infectious diseases}, number = {9}, publisher = {MDPI}, title = {{A structural perspective of the role of IP6 in immature and mature retroviral assembly}}, doi = {10.3390/v13091853}, volume = {13}, year = {2021}, } @article{9431, abstract = {Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.}, author = {Obr, Martin and Ricana, Clifton L. and Nikulin, Nadia and Feathers, Jon-Philip R. and Klanschnig, Marco and Thader, Andreas and Johnson, Marc C. and Vogt, Volker M. and Schur, Florian KM and Dick, Robert A.}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology, General Physics and Astronomy, General Chemistry}, number = {1}, publisher = {Nature Research}, title = {{Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer}}, doi = {10.1038/s41467-021-23506-0}, volume = {12}, year = {2021}, } @article{7464, abstract = {Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.}, author = {Dick, Robert A. and Xu, Chaoyi and Morado, Dustin R. and Kravchuk, Vladyslav and Ricana, Clifton L. and Lyddon, Terri D. and Broad, Arianna M. and Feathers, J. Ryan and Johnson, Marc C. and Vogt, Volker M. and Perilla, Juan R. and Briggs, John A. G. and Schur, Florian KM}, issn = {1553-7374}, journal = {PLOS Pathogens}, number = {1}, publisher = {Public Library of Science}, title = {{Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly}}, doi = {10.1371/journal.ppat.1008277}, volume = {16}, year = {2020}, }