Unbiased cleavage site prediction uncovers viral antagonism of host innate immunity by SARS-CoV-2 3C-like protease.

How SARS-CoV-2 causes a wide range of clinical manifestations and disease severity remains poorly understood. SARS-CoV-2 encodes 2 proteases (3CLPro and PLPro), vital for viral production, but also promiscuous with respect to host protein targets. Pharmacological inhibition of 3CLPro markedly reduced hospitalization and death in Phase 2/3 clinical studies. Here, we develop a bioinformatic algorithm, leveraging experimental data from SARS-CoV, to predict host cleavage targets of 3CLPro. We capture targets of 3CLPro described previously for SARS-CoV-2, as well as thousands of putative targets. We validate numerous targets cleaved during infection, including the giant sarcomeric protein obscurin and the innate immune protein OAS1. A long form of OAS1, p46, has been associated in numerous GWAS studies with lesser COVID disease severity. We show that 3CLPro cleaves p46 OAS1 immediately upstream of a known prenylation domain, relocalizing OAS1 from subcellular membranes to the cytosol, rendering it akin to the nonprotective, cytosolic p42 isoform. Similar OAS1 relocalization occurs upon infection by SARS-CoV-2. Our data provide a high-throughput resource to identify putative host cleavage targets of 3CLPro and reveal a mechanism by which SARS-CoV-2 antagonizes host innate immunity in individuals with the protective p46 isoform of OAS1.