Major genetic marker of nidoviruses encodes a replicative endoribonuclease

Abstract

Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the Ϸ30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn 2؉ -dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that doublestranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2-3 cyclic phosphate ends. 2-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription. H uman coronavirus 229E (HCoV-229E), a group 1 coronavirus, is one of the major viral pathogens causing upper respiratory tract illness in humans (1), whereas severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2 coronavirus, has been identified as the causative agent of SARS, a life-threatening form of pneumonia that caused Ͼ8,000 fatalities in a worldwide epidemic in 2003 (2, 3). The extremely large positive-strand RNA genomes of HCoV-229E (27.3 kb) and SARS-CoV (29.7 kb) contain 8 and 14 ORFs, respectively (4, 5). The two most 5Ј-terminal ORFs, 1a and 1b, encode the major subunits of the replicative machinery, whereas the more downstream ORFs encode structural and virus-specific accessory proteins.Coronavirus gene expression involves a series of complex transcriptional, translational, and posttranslational regulatory mechanisms (6, 7). After receptor-mediated entry, two large replicative polyproteins, pp1a (Ͼ450 kDa) and pp1ab (Ͼ750 kDa), are translated from the genome RNA. The polyproteins are encoded by the replicase gene (Ͼ20,000 bases) that comprise ORFs 1a and 1b (Fig. 1A) (8). Expression of pp1ab involves ribosomal frameshifting into...

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…Because of the large size of CoV genomes, which until recently has limited the use of reverse genetics approaches, the activities and functions of the various enzymes in the CoV life cycle are only slowly beginning to emerge. Recently, a CoV endoribonuclease activity has been identified and shown to have a critical role in CoV replication and transcription (14,15). Here, we report that CoVs encode a second ribonucleolytic activity.…”

“…SARS-CoV nsp14 was expressed as a maltose-binding protein (MBP) fusion protein in E. coli and purified by amylose-affinity chromatography, an approach that previously has proven suitable for the expression of several SARS-CoV enzymes (15)(16)(17). A significant portion of the overexpressed MBP-nsp14 fusion protein could be obtained in a soluble form and was purified by using a single-step purification protocol (Fig.…”

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation

“…These precursors undergo proteolytic processing via two virally encoded proteinases to generate 16 mature proteins, nsp1 through nsp16. Probably most of the gene 1 proteins are important for viral RNA synthesis (5,(7)(8)(9)(10)(11). Some gene 1 proteins are predicted to have biological functions related to RNA synthesis (11), and some have demonstrated functions that appear to be involved in RNA synthesis (7,8,10,12), whereas some gene 1 proteins may have functions other than viral RNA synthesis (13)(14)(15)(16).…”

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

“…Nsp15 has critical functions in viral replication and transcription, although how nsp15 acts in the viral cycle remains unclear (Bhardwaj et al, 2004;Ivanov et al, 2004). A mutation in nsp15 of HCoV-229E has been reported to prevent viral RNA accumulation, suggesting that nsp15 is (Ivanov et al, 2004). Mutations in the nsp15 orthologue of the related Arterivirus also prevented infections and reduced subgenomic RNA accumulation (Posthuma et al, 2006).…”

“…Nsp15, the only endoribonuclease residing in the genome, is considered to be a major 'genetic marker' of nidoviruses as it is not found in other RNA viruses (Ivanov et al, 2004). Nsp15 has critical functions in viral replication and transcription, although how nsp15 acts in the viral cycle remains unclear (Bhardwaj et al, 2004;Ivanov et al, 2004).…”