Significance We report an intersubunit interaction within the coronavirus replication–transcription complex that is critical for replication and evolutionarily conserved. We provide evidence that the nsp12-associated NiRAN domain has nucleoside monophosphate (NMP) transferase activity in trans and identified nsp9, an RNA-binding protein, as its target. NiRAN catalyzes the covalent attachment of an NMP moiety to the conserved nsp9 amino terminus in a reaction dependent on Mn 2+ ions and an adjacent conserved Asn residue. NiRAN activity and nsp9 NMPylation were found to be essential for coronavirus replication. The data lead us to connect this activity of a nidovirus enzymatic marker with previous observations within a functionally and evolutionarily coherent hypothesis on the initiation of RNA synthesis in a class of RNA viruses. 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 (M pro ). 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 Mn 2+ -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 M pro -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.
【저자키워드】 coronavirus, RNA polymerase, NiRAN, nidovirus, nucleotidyltransferase, 【초록키워드】 Nsp12, protease, in vitro, antiviral drug, activity, Replication, Protein, RNA viruses, Cell culture, Nsp9, RNA-dependent RNA polymerase, HCoV-229E, virus replication, mutant, Coronaviridae, Critical, marker, cellular, Evidence, nucleotide, Interaction, Hypothesis, Coronavirus replication, Nsps, cytidine, observation, acute respiratory syndrome, RNA synthesis, complex, residue, domain, M pro, sequence, transfer, family Coronaviridae, monophosphate, homolog, N-terminal, polyprotein, arterivirus, N terminus, nsp12 protein, RdRps, shown, virus, conserved, reported, generate, functional, provide, dependent on, suggested, released, Nidovirale, Ion, proteolytic, Significance, transferase activity, 【제목키워드】 Nsp9, subunit, selective, conserved, Vital,