Abstract
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.
Highlights
Positive-strand RNA viruses of the order Nidovirales infect diverse vertebrates and invertebrates [1, 2]
We report an intersubunit interaction within the coronavirus replication–transcription complex that is critical for replication and evolutionarily conserved
The two-domain combination of NiRAN-RNA-dependent RNA polymerases (RdRps) makes up nsp12 in the family Coronaviridae and nsp9 in the family Arteriviridae, and in other nidovirus families, NiRAN-RdRp is expected to be released as a separate nsp from the viral polyprotein(s)
Summary
Positive-strand RNA viruses of the order Nidovirales infect diverse vertebrates and invertebrates [1, 2]. In a final set of experiments, we produced C-terminally His6-tagged forms of SARS-CoV-2 nsp12 and nsp9 in E. coli, along with two mutant forms of nsp12 in which an active-site residue in the NiRAN and RdRp domain, respectively, was substituted with Ala (Fig. 6A and SI Appendix, Table S2).
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