Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor against the SARS-CoV-2 protease 3CL pro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein G, the SARS-CoV-2 3CL pro , and the VSV polymerase L. Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CL pro and release of the functional viral polymerase L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in an additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

中文翻译：

---

基于 VSV 的系统中选择的 SARS-CoV-2 3CL 原突变赋予对 nirmatrelvir、ensitrelvir 和 GC376 的耐药性

蛋白酶抑制剂是最有效的抗病毒药物之一。Nirmatrelvir 是第一个针对 SARS-CoV-2 蛋白酶 3CL 的蛋白酶抑制剂亲已获得临床使用许可。为了识别赋予这种蛋白酶抑制剂抗性的突变，我们设计了一种嵌合水泡性口炎病毒（VSV），它表达由 VSV 糖蛋白 G 组成的多蛋白，即 SARS-CoV-2 3CL亲，和 VSV 聚合酶 L。因此，病毒复制依赖于 3CL 对该前体蛋白的自催化加工亲和功能性病毒聚合酶 L 的释放，并且该嵌合 VSV 的复制被 nirmatrelvir 有效抑制。使用该系统，我们应用 nirmatrelvir 来选择耐药突变。通过在另外的基于 VSV 的系统、独立开发的细胞系统、生化检测和重组 SARS-CoV-2 系统中重新测试 nirmatrelvir 针对选定突变的耐药性得到确认。我们证明一些突变体对 ensitrelvir 和 GC376 具有交叉耐药性，而其他突变体对这些化合物的耐药性较低。此外，我们发现大多数耐药突变已经存在于 NCBI 和 GISAID 数据库中的 SARS-CoV-2 序列中，表明这些突变存在于流通的 SARS-CoV-2 毒株中。