Biology subjects

Winter, S. L.

Publications and source records attributed to Winter, S. L..

6 recordsLinked to original sources

bioRxiv2023

Nucleocapsid condensation drives Ebola viral factory maturation and dispersion

Replication and genome encapsidation of many negative-sense RNA viruses take place in virus-induced membrane-less organelles termed viral factories (VFs). While liquid properties of VFs are believed to control the transition from genome replication to encapsidation, the nucleocapsid assembly, VF maturation and interactions with the cellular environment remain elusive. Here we apply in situ cryo-correlative light and electron tomography to follow nucleocapsid assembly and changes in VF morphology and their liquid properties during Ebola virus infection. We show that Ebola viral nucleocapsids transition from loosely packed helical assemblies in early VFs to condensed cylinders that arrange into highly organized parallel bundles later in infection. Early VFs associate with intermediate filaments and are devoid of other host material, but become progressively accessible to cellular components. Our data suggest that this process is coupled to VF solidification and dispersion, and that changes in liquid properties of VFs promote nucleocapsid transport to budding sites. Highlights- Cryo-ET reveals the molecular architecture of Ebola virus replication compartments - Loosely coiled nucleocapsids transition to condensed cylinders forming bundles - Nucleocapsid condensation drives dispersion of viral factories promoting viral egress - Intermediate filaments associate with and are critical for virus factory formation

microbiology↗

bioRxiv2022

The Ebola virus VP40 matrix undergoes endosomal disassembly essential for membrane fusion

Ebola viruses (EBOVs) are filamentous particles, whose shape and stability are determined by the VP40 matrix. Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

microbiology↗

bioRxiv2022

The glycolipid GlcCer is recruited into the viral envelope to promote phenuivirus binding to host cells

Virus-receptor interactions largely contribute to the tropism and outcome of an infection. Here, we found that the glycolipid glucosylceramide (GlcCer) is a major component of Uukuniemi phenuivirus and allows viral binding to host cells. A lipidomic analysis with mass spectrometry revealed the lipidome of UUKV particles and indicated that GlcCer was enriched in both infected cells and viral particles. In addition, the infectivity of UUKV depended on the conversion of ceramide (Cer) into GlcCer in the Golgi network of producer cells. In contrast, depletion of GlcCer in virions profoundly impaired the attachment of UUKV and other related viruses to target cells. Furthermore, competing GlcCer ligands prevented virus binding to various cell types. Altogether, our results demonstrate that glycolipids are essential structural determinants of the virions necessary for virus attachment to host cells and have strong implications for future work on the identification of virus receptors.

microbiology↗

bioRxiv2021

Dual-axis Volta phase plate cryo-electron tomography of Ebola virus-like particles reveals actin-VP40 interactions

Cryo-electron tomography (cryo-ET) is a pivotal imaging technique for studying the structure of pleomorphic enveloped viruses and their interactions with the host at native conditions. Owing to the limited tilting range of samples with a slab geometry, electron tomograms suffer from so-called missing wedge information in Fourier space. In dual-axis cryo-ET, two tomograms reconstructed from orthogonally oriented tilt series are combined into a tomogram with improved resolution as the missing wedge information is reduced to a pyramid. Volta phase plate (VPP) allows to perform in-focus cryo-ET with high contrast transfer at low-resolution frequencies and thus its application may improve the quality of dual-axis tomograms. Here, we compare dual-axis cryo-ET with and without VPP on Ebola virus-like particles to visualize and segment viral and host cell proteins within the membrane-enveloped filamentous particles. Dual-axis VPP cryo-ET reduces the missing wedge information and ray artifacts arising from the weighted back-projection during tomogram reconstruction, thereby minimizing ambiguity in the analysis of crowded environments and facilitating 3D segmentation. We show that dual-axis VPP tomograms provide a comprehensive description of macromolecular organizations such as nucleocapsid assembly states, the distribution of glycoproteins on the viral envelope and asymmetric arrangements of the VP40 layer in non-filamentous regions of virus-like particles. Our data reveal actin filaments within virus-like particles in close proximity to the viral VP40 scaffold, suggesting a direct interaction between VP40 and actin filaments. Dual-axis VPP cryo-ET provides more complete 3D information at high contrast and allows for better interpretation of macromolecule interactions and pleomorphic organizations. HighlightsO_LIVolta phase plate dual-axis cryo-electron tomography provides high contrast tomography data with reduced back-projection ray artifacts and missing wedge information in Fourier space C_LIO_LIVolta phase plate dual-axis cryo-electron tomography facilitates interpretation of protein-membrane interactions C_LIO_LIVolta phase plate dual-axis cryo-electron tomography reduces ambiguity in manual 3D rendering and markedly improves 3D isosurface modeling C_LIO_LIEbola virus-like particles contain actin filaments in close proximity to the VP40 layer C_LI

biophysics↗

bioRxiv2020

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic {beta}-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

microbiology↗

bioRxiv2020

Membrane architecture of pulmonary lamellar bodies revealed by post-correlation on-lamella cryo-CLEM

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through "T"-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

cell biology↗