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Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

View ORCID ProfileBjoern Meyer, View ORCID ProfileJeanne Chiaravalli, View ORCID ProfileStacy Gellenoncourt, View ORCID ProfilePhilip Brownridge, View ORCID ProfileDominic P. Bryne, View ORCID ProfileLeonard A. Daly, View ORCID ProfileMarius Walter, View ORCID ProfileFabrice Agou, View ORCID ProfileLisa A. Chakrabarti, View ORCID ProfileCharles S. Craik, View ORCID ProfileClaire E. Eyers, View ORCID ProfilePatrick A. Eyers, View ORCID ProfileYann Gambin, View ORCID ProfileEmma Sierecki, View ORCID ProfileEric Verdin, View ORCID ProfileMarco Vignuzzi, View ORCID ProfileEdward Emmott
doi: https://doi.org/10.1101/2020.09.16.297945
Bjoern Meyer
1Viral Populations and Pathogenesis Unit, CNRS, UMR 3569, Institut Pasteur, Paris, CEDEX 15, France
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Jeanne Chiaravalli
2Chemogenomic and Biological Screening Core Facility, C2RT, Departments of Cell Biology & Infection and of Structural Biology Chemistry, Institut Pasteur, Paris, CEDEX 15, France
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Stacy Gellenoncourt
3CIVIC Group, Virus Immunity Unit, Institut Pasteur and CNRS UMR 3569, Paris, France
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Philip Brownridge
4Centre for Proteome Research, Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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Dominic P. Bryne
5Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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Leonard A. Daly
4Centre for Proteome Research, Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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Marius Walter
6Buck Institute for Research on Aging, Novato, CA, 94945, USA
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Fabrice Agou
2Chemogenomic and Biological Screening Core Facility, C2RT, Departments of Cell Biology & Infection and of Structural Biology Chemistry, Institut Pasteur, Paris, CEDEX 15, France
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Lisa A. Chakrabarti
3CIVIC Group, Virus Immunity Unit, Institut Pasteur and CNRS UMR 3569, Paris, France
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Charles S. Craik
7Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA
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Claire E. Eyers
4Centre for Proteome Research, Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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Patrick A. Eyers
5Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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Yann Gambin
8EMBL Australia Node for Single Molecule Sciences, and School of Medical Sciences, Botany Road, The University of New South Wales, Sydney NSW 2052, Australia
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Emma Sierecki
8EMBL Australia Node for Single Molecule Sciences, and School of Medical Sciences, Botany Road, The University of New South Wales, Sydney NSW 2052, Australia
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Eric Verdin
6Buck Institute for Research on Aging, Novato, CA, 94945, USA
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Marco Vignuzzi
1Viral Populations and Pathogenesis Unit, CNRS, UMR 3569, Institut Pasteur, Paris, CEDEX 15, France
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Edward Emmott
4Centre for Proteome Research, Department of Biochemistry & Systems Biology, Institute of Systems, Molecular & Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool, L69 7ZB, UK
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  • For correspondence: e.emmott@liverpool.ac.uk
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Abstract

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 100 million infections, and over 2 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19 disease.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Overall the manuscript has been revised with additional independent validation of cellular cleavage in vitro and in cell-based assays of cellular substrates identified by N-terminomics. Additionally, further N-terminomics experiments have identified the likely causal proteases of a subset of novel viral cleavage sites, and shown that mutations proximal to our 637 cleavage site in spike alter cell entry and cleavage state in pseudotyped lentivirus.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted February 05, 2021.
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Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing
Bjoern Meyer, Jeanne Chiaravalli, Stacy Gellenoncourt, Philip Brownridge, Dominic P. Bryne, Leonard A. Daly, Marius Walter, Fabrice Agou, Lisa A. Chakrabarti, Charles S. Craik, Claire E. Eyers, Patrick A. Eyers, Yann Gambin, Emma Sierecki, Eric Verdin, Marco Vignuzzi, Edward Emmott
bioRxiv 2020.09.16.297945; doi: https://doi.org/10.1101/2020.09.16.297945
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Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing
Bjoern Meyer, Jeanne Chiaravalli, Stacy Gellenoncourt, Philip Brownridge, Dominic P. Bryne, Leonard A. Daly, Marius Walter, Fabrice Agou, Lisa A. Chakrabarti, Charles S. Craik, Claire E. Eyers, Patrick A. Eyers, Yann Gambin, Emma Sierecki, Eric Verdin, Marco Vignuzzi, Edward Emmott
bioRxiv 2020.09.16.297945; doi: https://doi.org/10.1101/2020.09.16.297945

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