The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNAdependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positivestrand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between 1EV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in 11EV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.Hepatitis E virus (HEV) is the causative agent of the enterically transmitted form of non-A, non-B hepatitis, a disease of significant epidemiologic importance (1, 2). HEV has been found in stools from hepatitis patients as naked isometric virus-like particles of about 32-34 nm in diameter (3, 4). It has been shown that the HEV genome is a single-stranded polyadenylylated RNA of about 7.5 kilobases (5). Recently the HEV genome has been cloned as cDNA (5, 6), and its complete nucleotide sequence has been determined (7,8). The positive-sense RNA of HEV encompasses three large open reading frames (ORFs): the largest ORF (5' end) consists of 1693 codons, the second ORF (3' end) is composed of 660 codons, and the third ORF consists of 123 codons that overlap the first and the second ORFs. By analogy with other positive-strand RNA animal viruses, such as alphaviruses (9), rubella virus (RubV), (10) and caliciviruses (11), it was tempting to speculate that the 660-codon ORF encodes the capsid protein(s) of HEV, whereas the product of the largest 5' ORF should be the nonstructural polyprotein, which is probably cleaved to yield functional viral proteins (7,8). The function of the smallest ORF is not known, but there are indications that it is expressed in infected humans (12).Comparative analyses of the sequences of nonstructural proteins encoded by positive-strand RNA viruses have yielded a considerable collection of functional domains with varying degrees of conservation. Various subsets of this "pool" are combined in polyproteins (or large ...

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...

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