A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

IF 6.7 1区 医学 Q1 Immunology and Microbiology PLoS Pathogens Pub Date : 2023-08-31 eCollection Date: 2023-08-01 DOI:10.1371/journal.ppat.1011614
Taha Y Taha, Rahul K Suryawanshi, Irene P Chen, Galen J Correy, Maria McCavitt-Malvido, Patrick C O'Leary, Manasi P Jogalekar, Morgan E Diolaiti, Gabriella R Kimmerly, Chia-Lin Tsou, Ronnie Gascon, Mauricio Montano, Luis Martinez-Sobrido, Nevan J Krogan, Alan Ashworth, James S Fraser, Melanie Ott
{"title":"A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.","authors":"Taha Y Taha, Rahul K Suryawanshi, Irene P Chen, Galen J Correy, Maria McCavitt-Malvido, Patrick C O'Leary, Manasi P Jogalekar, Morgan E Diolaiti, Gabriella R Kimmerly, Chia-Lin Tsou, Ronnie Gascon, Mauricio Montano, Luis Martinez-Sobrido, Nevan J Krogan, Alan Ashworth, James S Fraser, Melanie Ott","doi":"10.1371/journal.ppat.1011614","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":20178,"journal":{"name":"PLoS Pathogens","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2023-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10499221/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"PLoS Pathogens","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1371/journal.ppat.1011614","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/8/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"Immunology and Microbiology","Score":null,"Total":0}
引用次数: 0

Abstract

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.

Abstract Image

Abstract Image

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
严重急性呼吸系统综合征冠状病毒2型NSP3 Mac1结构域中的单一失活氨基酸变化减弱了病毒在体内的复制。
尽管做出了前所未有的努力,但我们对抗严重急性呼吸系统综合征冠状病毒2型的治疗手段仍然有限。NSP3中保守的大结构域1(Mac1)是一种表现出ADP核糖水解酶活性的酶,也是一种可能的药物靶点。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,产生了编码催化失活的NSP3-Mac1结构域的重组病毒和复制子。与野生型相比,丙氨酸(N40A)的取代使催化活性降低了约10倍,天冬氨酸(N40D)的突变使催化活性减少了约100倍。重要的是,N40A突变使Mac1在体外不稳定,并降低了细菌和哺乳动物细胞中的表达水平。当纳入严重急性呼吸系统综合征冠状病毒2型分子克隆时,N40D突变体仅适度影响永生细胞系中的病毒适应度,但将病毒在人类气道类器官中的复制减少了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,同时诱导了强大的干扰素反应;所有感染变异病毒的动物都存活了下来。我们的数据验证了严重急性呼吸系统综合征冠状病毒2型NSP3 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一个有前景的治疗靶点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
PLoS Pathogens
PLoS Pathogens 生物-病毒学
CiteScore
11.40
自引率
3.00%
发文量
598
审稿时长
2 months
期刊介绍: Bacteria, fungi, parasites, prions and viruses cause a plethora of diseases that have important medical, agricultural, and economic consequences. Moreover, the study of microbes continues to provide novel insights into such fundamental processes as the molecular basis of cellular and organismal function.
期刊最新文献
Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection Turning the needle into the haystack: Culture-independent amplification of complex microbial genomes directly from their native environment Drivers of diversification in fungal pathogen populations α-Synuclein strain propagation is independent of cellular prion protein expression in a transgenic synucleinopathy mouse model A comprehensive study of SARS-CoV-2 mfigain protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1