尽管付出了前所未有的努力，我们对抗SARS-CoV-2的治疗武器仍然有限。NSP3中保守的巨噬细胞结域1（Mac1）是一种表现出ADP-核糖水解酶活性的酶，也是一个可能的药物靶点。为了确定Mac1催化活性在病毒复制中的作用，我们通过突变活性位点中的关键天冬酰胺，在重组病毒和复制子中编码一个催化不活性的NSP3 Mac1结构域。虽然丙氨酸（N40A）的替换使催化活性降低约10倍，而门冬氨酸（N40D）的突变使活性相对于野生型降低了约100倍。重要的是，N40A突变使Mac1在体外不稳定，并降低了在细菌和哺乳动物细胞中的表达水平。当纳入SARS-CoV-2分子克隆体中时，N40D突变体只对不死细胞株的病毒适应性产生轻微影响，但将人类气道器官样细胞内的病毒复制降低了10倍。在小鼠中，与野生型病毒相比，N40D突变体的复制水平降低了1000倍以上，同时诱导了强烈的干扰素应答；所有被突变病毒感染的动物均存活下来。我们的数据验证了SARS-CoV-2 NSP3 Mac1催化活性在病毒复制中的关键作用，并作为一个有前景的治疗靶点来开发抗病毒药物。版权所有：©2023 Taha等人。本文系按照创作共用署名许可协议（Creative Commons Attribution License）进行开放访问分发的文章，允许在任何媒体中进行无限制的使用、分发和复制，只要保留原作者和来源的署名。

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.Copyright: © 2023 Taha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.