Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites

The SARS-CoV-2 3CL protease (3CL^pro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CL^pro and validate a subset of our results within authentic viruses. We reveal that the 3CL^pro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CL^pro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CL^pro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CL^pro and for drug development against coronaviruses.