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.

SARS-CoV-2 3CL 蛋白酶（3CL ^pro）是一个有吸引力的治疗靶点，因为它对病毒至关重要，并且在冠状病毒中高度保守。然而，我们目前对其突变耐受性的了解是有限的。^{在这里，我们开发了一种基于酵母的深度突变扫描方法，以系统地分析 3CL pro}所有可能的单突变体的活性，并在真实病毒中验证我们的结果的子集。我们发现 3CL ^pro具有高度可塑性，并且能够耐受整个蛋白质的突变。然而，我们还鉴定出了看似不可变的特定残基，表明这些残基可能是未来 3CL^前体抑制剂的目标。^{最后，我们利用我们的筛选作为基础，将 E166V 鉴定为针对临床使用的 3CL前}抑制剂 nirmatrelvir 的耐药性突变。总的来说，本文提出的功能图谱可以作为更好地了解 3CL ^pro的生物学特性和针对冠状病毒的药物开发的指南。