Identification of Human Host Substrates of the SARS-CoV-2 M^pro and PL^pro Using Subtiligase N-Terminomics

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, M^pro and PL^pro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 M^pro and PL^pro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 M^pro and PL^pro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.