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SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging

Luiza Mendonça, Andrew Howe, James B. Gilchrist, Dapeng Sun, Michael L. Knight, Laura C. Zanetti-Domingues, Benji Bateman, Anna-Sophia Krebs, Long Chen, Julika Radecke, Yuewen Sheng, Vivian D. Li, Tao Ni, Ilias Kounatidis, Mohamed A. Koronfel, Marta Szynkiewicz, View ORCID ProfileMaria Harkiolaki, Marisa L. Martin-Fernandez, William James, View ORCID ProfilePeijun Zhang
doi: https://doi.org/10.1101/2020.11.05.370239
Luiza Mendonça
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Andrew Howe
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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James B. Gilchrist
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Dapeng Sun
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Michael L. Knight
3Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK
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Laura C. Zanetti-Domingues
4Central Laser Facility, Science and Technology Facility Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire. OX11 0FA, UK
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Benji Bateman
4Central Laser Facility, Science and Technology Facility Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire. OX11 0FA, UK
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Anna-Sophia Krebs
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Long Chen
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Julika Radecke
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Yuewen Sheng
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Vivian D. Li
5Murray Edwards College, University of Cambridge, Cambridge, CB3 0DF, UK
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Tao Ni
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Ilias Kounatidis
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Mohamed A. Koronfel
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Marta Szynkiewicz
4Central Laser Facility, Science and Technology Facility Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire. OX11 0FA, UK
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Maria Harkiolaki
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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  • ORCID record for Maria Harkiolaki
Marisa L. Martin-Fernandez
4Central Laser Facility, Science and Technology Facility Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire. OX11 0FA, UK
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William James
3Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK
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Peijun Zhang
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
2Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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  • ORCID record for Peijun Zhang
  • For correspondence: peijun@strubi.ox.ac.uk
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Summary

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

Competing Interest Statement

The authors have declared no competing interest.

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SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging
Luiza Mendonça, Andrew Howe, James B. Gilchrist, Dapeng Sun, Michael L. Knight, Laura C. Zanetti-Domingues, Benji Bateman, Anna-Sophia Krebs, Long Chen, Julika Radecke, Yuewen Sheng, Vivian D. Li, Tao Ni, Ilias Kounatidis, Mohamed A. Koronfel, Marta Szynkiewicz, Maria Harkiolaki, Marisa L. Martin-Fernandez, William James, Peijun Zhang
bioRxiv 2020.11.05.370239; doi: https://doi.org/10.1101/2020.11.05.370239
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SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging
Luiza Mendonça, Andrew Howe, James B. Gilchrist, Dapeng Sun, Michael L. Knight, Laura C. Zanetti-Domingues, Benji Bateman, Anna-Sophia Krebs, Long Chen, Julika Radecke, Yuewen Sheng, Vivian D. Li, Tao Ni, Ilias Kounatidis, Mohamed A. Koronfel, Marta Szynkiewicz, Maria Harkiolaki, Marisa L. Martin-Fernandez, William James, Peijun Zhang
bioRxiv 2020.11.05.370239; doi: https://doi.org/10.1101/2020.11.05.370239

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