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Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Article in English | MEDLINE | ID: covidwho-1039676


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.

Coronavirus 229E, Human/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication , Amino Acid Sequence , Amino Acid Substitution , Asparagine/genetics , Cell Line , Conserved Sequence , Coronavirus 229E, Human/physiology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Manganese/metabolism , Protein Domains , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription, Genetic , Viral Nonstructural Proteins/genetics
Biochem Biophys Res Commun ; 538: 24-34, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-921830


Two pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st century raised unprecedented interest in coronavirus research and assigned it unseen urgency. The two viruses responsible for the outbreaks, SARS-CoV and SARS-CoV-2, respectively, are in the spotlight, and SARS-CoV-2 is the focus of the current fast-paced research. Its foundation was laid down by studies of many corona- and related viruses that collectively form the vast order Nidovirales. Comparative genomics of nidoviruses played a key role in this advancement over more than 30 years. It facilitated the transfer of knowledge from characterized to newly identified viruses, including SARS-CoV and SARS-CoV-2, as well as contributed to the dissection of the nidovirus proteome and identification of patterns of variations between different taxonomic groups, from species to families. This review revisits selected cases of protein conservation and variation that define nidoviruses, illustrates the remarkable plasticity of the proteome during nidovirus adaptation, and asks questions at the interface of the proteome and processes that are vital for nidovirus reproduction and could inform the ongoing research of SARS-CoV-2.

Coronaviridae Infections/virology , Nidovirales/classification , Nidovirales/genetics , Conserved Sequence , Evolution, Molecular , Genetic Variation , Genomics , Humans , Phylogeny , Proteome , SARS Virus/classification , SARS Virus/genetics , SARS-CoV-2/classification , SARS-CoV-2/genetics , Viral Proteins