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Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
Keywords
SARS-CoV-2, COVID-19, cryoEM, cryoET, subtomogram averaging, cryoFIB/SEM, soft X-ray cryo-tomography, virus assembly, egress, spike
Figure 1
Figure 2
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Figure 4
Figure 5
Figure 6
There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- SARSCoV2MultiScaleSIFigures12142020.pdf
Supplementary Figures
- Movie1cell8annotatedfullres.avi
Serial cryoFIB/SEM of an uninfected cell
- Movie2cell1annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell
- Movie3Cell1segmentation.mp4
Segmentation of the volume depicted in Movie 2
- Movie4cell5annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell, showing abound heterogeneous vesicles
- Movie5cell3annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell, showing cytoplasmic invagination
- Movie6LM443SIC1lamella008.mp4
CryoET of DMVs and portals
- Movie7LM422SIC1lamellatomo005.mp4
CryoET of virus assembly sites
- Movie8LM442SIC1027.mp4
CryoET of released viruses
- Movie9LM433SIC1Au002.mp4
CryoET of virus egress site, broken membrane
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Nature Communications.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Article
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Nature Communications.
Version 1
Posted 19 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Nature Communications.
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- SARSCoV2MultiScaleSIFigures12142020.pdf
Supplementary Figures
- Movie1cell8annotatedfullres.avi
Serial cryoFIB/SEM of an uninfected cell
- Movie2cell1annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell
- Movie3Cell1segmentation.mp4
Segmentation of the volume depicted in Movie 2
- Movie4cell5annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell, showing abound heterogeneous vesicles
- Movie5cell3annotatedfullres.mp4
Serial cryoFIB/SEM of an infected cell, showing cytoplasmic invagination
- Movie6LM443SIC1lamella008.mp4
CryoET of DMVs and portals
- Movie7LM422SIC1lamellatomo005.mp4
CryoET of virus assembly sites
- Movie8LM442SIC1027.mp4
CryoET of released viruses
- Movie9LM433SIC1Au002.mp4
CryoET of virus egress site, broken membrane
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