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

Winter, Sophie L.; Chlanda, Petr

doi:10.1016/j.jsb.2021.107742

Cited by 19 publications

(11 citation statements)

References 49 publications

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“…Ebola virus VLPs and chemically fixed EBOV were plunge‐frozen as previously described (Winter & Chlanda, 2021 ). Briefly, VLPs were diluted to approximately 10–20 ng/μl, mixed with 10 nm protein A‐coated colloidal gold (Aurion), and applied onto glow‐discharged EM grids (200 mesh, R 2/1, Quantifoil) prior to plunge‐freezing with a Leica EM GP2 automatic plunge‐freezer.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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“…Examples abound, ranging from pleomorphic viruses 3 to cellular organelles 4 , to large-scale cellular structures like synapses between neurons 5 , 6 . Many viruses, notably those involved in devastating pandemics such as SARS-CoV-2 7 – 9 , influenza viruses 10 , and human immunodeficiency viruses (HIV) 11 , are pleomorphic in the organizations of their proteins and genomes. Cellular organelles, such as axonemes containing microtubule doublets surrounding a central pair 12 , though largely conserved in their core elements across different species, rely on their non-conserved and variable attachment of peripheral components that define their characteristic species-specific functions 13 .…”

Section: Introductionmentioning

confidence: 99%

Isotropic reconstruction for electron tomography with deep learning

et al. 2022

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Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic “missing-wedge” problem has presented major challenges in visualization and interpretation of tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs the missing-wedge information and increases signal-to-noise ratio, using the knowledge learned from raw tomograms. Without the need for sub-tomogram averaging, IsoNet generates tomograms with significantly reduced resolution anisotropy. Applications of IsoNet to three representative types of cryoET data demonstrate greatly improved structural interpretability: resolving lattice defects in immature HIV particles, establishing architecture of the paraflagellar rod in Eukaryotic flagella, and identifying heptagon-containing clathrin cages inside a neuronal synapse of cultured cells. Therefore, by overcoming two fundamental limitations of cryoET, IsoNet enables functional interpretation of cellular tomograms without sub-tomogram averaging. Its application to high-resolution cellular tomograms should also help identify differently oriented complexes of the same kind for sub-tomogram averaging.

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“…showed that VFs are exclusively composed of loosely coiled left-handed helical structures (Fig. 2A-F) resembling those that assemble in vitro upon expression of NP and represent the precursor to the fully assembled EBOV NC [29, 30]. The results could be confirmed by cryo-ET analysis of cells infected with wildtype EBOV fixed at 14 h p.i.…”

Section: Resultsmentioning

confidence: 70%

Nucleocapsid condensation drives Ebola viral factory maturation and dispersion

Vallbracht,

Bodmer,

Fischer

et al. 2023

Preprint

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SummaryReplication 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 applyin situcryo-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

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

Cited by 19 publications

References 49 publications

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

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

Isotropic reconstruction for electron tomography with deep learning

Nucleocapsid condensation drives Ebola viral factory maturation and dispersion

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