气候变化领域集成服务门户(CCPortal): Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit

CCPortal

DOI	10.1073/pnas.2022310118
	Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit
	Slanina H.; Madhugiri R.; Bylapudi G.; Schultheiß K.; Karl N.; Gulyaeva A.; Gorbalenya A.E.; Linne U.; Ziebuhr J.
发表日期	2021
ISSN	00278424
卷号	118 期号:6
英文摘要	RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae. The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development. © This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).
英文关键词	Coronavirus; Nidovirus; NiRAN; Nucleotidyltransferase; RNA polymerase
语种	英语
scopus关键词	asparagine; manganese; NSP12 protein, SARS-CoV-2; NSP9 protein, SARS-CoV-2; recombinant protein; RNA binding protein; viral protein; amino acid sequence; amino acid substitution; cell line; conserved sequence; genetic transcription; genetics; human; Human coronavirus 229E; metabolism; physiology; protein domain; virus replication; Amino Acid Sequence; Amino Acid Substitution; Asparagine; Cell Line; Conserved Sequence; Coronavirus 229E, Human; Coronavirus RNA-Dependent RNA Polymerase; Humans; Manganese; Protein Domains; Recombinant Proteins; RNA-Binding Proteins; SARS-CoV-2; Transcription, Genetic; Viral Nonstructural Proteins; Virus Replication
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180781
作者单位	Institute of Medical Virology, Justus Liebig University Giessen, Giessen, 35392, Germany; Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Netherlands; Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, 2300 RC, Netherlands; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119991, Russian Federation; Mass Spectrometry Facility, Department of Chemistry, Philipps University Marburg, Marburg, 35043, Germany; Department of Genetics, University Medical Center Groningen, Groningen, 9713 GZ, Netherlands
推荐引用方式 GB/T 7714	Slanina H.,Madhugiri R.,Bylapudi G.,et al. Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit[J],2021,118(6).
APA	Slanina H..,Madhugiri R..,Bylapudi G..,Schultheiß K..,Karl N..,...&Ziebuhr J..(2021).Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit.Proceedings of the National Academy of Sciences of the United States of America,118(6).
MLA	Slanina H.,et al."Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit".Proceedings of the National Academy of Sciences of the United States of America 118.6(2021).