A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.
A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.
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DOI：
10.1371/journal.ppat.1011614
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发表时间：
2023-08
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
尽管作出了前所未有的努力，但我们对抗SARS-CoV-2的治疗武器仍然有限。NSP3中保守的大结构域1(MAC1)是一种显示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，我们通过突变活性部位的关键天冬酰胺来产生编码NSP3 MAC1结构域的重组病毒和复制子。当突变为丙氨酸(N40A)时，催化活性降低约10倍，而突变为天冬氨酸(N40D)时，活性降低约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。当被整合到SARS-CoV-2分子克隆中时，N40D突变体只对永生化细胞系中的病毒适合性产生了轻微影响，但将病毒在人类呼吸道器官中的复制减少了10倍。在小鼠身上，N40D突变株的复制水平比野生型病毒低1000倍，同时诱导了强大的干扰素反应；所有感染突变病毒的动物都存活了下来。我们的数据证实了SARS-CoV-2NSP3 MAC1催化活性在病毒复制中的关键作用，并作为开发抗病毒药物的有前景的治疗靶点。冠状病毒病2019(新冠肺炎)大流行是由严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的，仍然是一个全球公共卫生问题。尽管我们在开发针对SARS-CoV-2的疫苗和疗法方面做出了前所未有的努力，但由于缺乏有效的病毒药物靶点，我们的治疗手段仍然局限于有限数量的疗法。在这里，我们确定了SARS-CoV-2中一个潜在的新药靶点。MAC1结构域是冠状病毒中一种进化上保守的酶，它可以去除蛋白质中ADP-核糖的翻译后修饰，从而对抗宿主的先天性免疫反应。我们用单点突变产生了SARS-CoV-2，使MAC1结构域催化失活，并证明了这种突变病毒不太适合迷你肺、碟子(有机物)和小鼠。这项工作为针对MAC1催化活性作为SARS-CoV-2抗病毒药物的新疗法的开发铺平了道路。
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. The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and remains a public health issue worldwide. Despite unprecedented efforts to develop vaccines and therapeutics against SARS-CoV-2, our therapeutic arsenal remains limited to only a limited number of therapeutics due to the lack of validated viral drug targets. Here, we identify a potentially new drug target in SARS-CoV-2. The Mac1 domain is an evolutionarily conserved enzyme in coronaviruses that can remove ADP-ribose post-translational modifications from proteins thereby combating host innate immune responses. We generated SARS-CoV-2 with a single point mutation that renders the Mac1 domain catalytically inactive and demonstrated that this mutant virus is less fit in mini lungs in the dish (organoids) and in mice. This work paves the way for the development of novel therapeutics targeting the Mac1 catalytic activity as SARS-CoV-2 antivirals.
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影响因子：
16
作者：
Kraus, W. Lee
通讯作者：
Kraus, W. Lee
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
30.3
作者：
Case, James Brett;Rothlauf, Paul W.;Whelan, Sean P. J.
通讯作者：
Whelan, Sean P. J.
影响因子：
2.1
作者：
Hoch, Nicolas C.;Polo, Luis M.
通讯作者：
Polo, Luis M.
DOI：
10.1056/nejmoa2118542
发表时间：
2022-04-14
期刊：
The New England journal of medicine
影响因子：
--
作者：
Hammond J;Leister-Tebbe H;Gardner A;Abreu P;Bao W;Wisemandle W;Baniecki M;Hendrick VM;Damle B;Simón-Campos A;Pypstra R;Rusnak JM;EPIC-HR Investigators
通讯作者：
EPIC-HR Investigators