Viral proteases play key roles in viral replication, and they also facilitate immune escape by proteolyzing diverse target proteins. Deep profiling of viral protease substrates in host cells is beneficial for understanding viral pathogenesis and for antiviral drug discovery. Here, we utilized substrate phage display coupled with protein network analysis (SPD-PNA) to identify human proteome substrates of SARS-CoV-2 viral proteases, including papain-like protease (PLpro) and 3C-like protease (3CLpro). We first performed peptide substrates selection of PLpro and 3CLpro, and we then used the top 24 preferred substrate sequences to identify a total of 290 putative protein substrates. Protein network analysis revealed that the top clusters of PLpro and 3CLpro substrate proteins contain ubiquitin-related proteins and cadherin-related proteins respectively. We verified that cadherin-6 (CDH6) and cadherin-12 (CDH12) are novel substrates of 3CLpro and CD177 is a novel substrate of PLpro using in vitro cleavage assays. We thus demonstrated SPD-PNA is a simple and high throughput method to identify human proteome substrates of SARS-CoV-2 viral proteases for further understanding of virus-host interactions.

Fuente: Journal of Biological Chemistry
Available online 16 May 2023