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 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 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.

Nidovirales（冠状病毒科，Arteriviridae和其他12个科）的RNA依赖性RNA聚合酶（RdRps）与从多蛋白释放的非结构蛋白（nsp）的氨基末端（N-末端）域相连，称为NiRAN。 1ab由病毒主要蛋白酶（M ^pro）。以前，据报道动脉病毒NiRAN-RdRp nsp具有自我GMPylation / UMPylation活性，并建议产生一个过渡状态，用于将核苷单磷酸酯（NMP）转移至（目前未知）病毒和/或细胞生物聚合物。在这里，我们显示冠状病毒（人类冠状病毒[HCoV] -229E和严重急性呼吸系统综合症冠状病毒2）nsp12（NiRAN-RdRp）具有Mn ²⁺依赖的NMPylation活性，在M ^pro之后，通过与nsp9 N末端（N3825）的伯胺形成氨基磷酸酯键，催化单个NMP向同源nsp9的转移介导的Nsp9从N末端侧翼nsps的蛋白水解释放。尿苷三磷酸是该反应中优选的核苷酸，但是腺苷三磷酸，鸟苷三磷酸和胞苷三磷酸也是合适的共底物。使用重组冠状病毒nsp9和nsp12蛋白以及经过基因工程改造的HCoV-229E突变体进行的突变研究确定了在细胞培养中NiRAN介导的nsp9 NMP化和病毒复制所必需的残基。数据证实了对NiRAN活性位点残基的预测，并确定了nsp9 N3826残基在nsp9 NMPylation体外和病毒复制中的重要作用。该残基是保守的N端NNE三肽序列的一部分，显示为nsp9及其冠状病毒科病毒中同源物的唯一不变残基。该研究为其他Nidovirus NMPylation活性的功能研究提供了坚实的基础，并提出了抗病毒药物开发的可能目标。