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.
