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.

No abstract

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37TC, suggesting the presence of a receptordestroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemag-utination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Closrtidimwperfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize 0-acetylated'sialic acid or a similar derivative as cell receptor.

Many positive-stranded RNA viruses use subgenomic mRNAs to express part of their genetic information. To produce structural and accessory proteins, members of the order Nidovirales (corona-, toro-, arteri-and roniviruses) generate a 39 co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 39 coterminal but also contain a common 59 leader sequence, which is derived from the genomic 59 end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarityassisted RNA recombination. Most models proposed over the past 25 years assume co-transcriptional fusion of subgenomic RNA leader and body sequences, but there has been controversy over the question of whether this occurs during plus-or minus-strand synthesis. In the latter model, which has now gained considerable support, subgenomic mRNA synthesis takes place from a complementary set of subgenome-size minus-strand RNAs, produced by discontinuous minus-strand synthesis. Sense-antisense base-pairing interactions between short conserved sequences play a key regulatory role in this process. In view of the presumed common ancestry of nidoviruses, the recent finding that ronivirus and torovirus mRNAs do not contain a common 59 leader sequence is surprising. Apparently, major mechanistic differences must exist between nidoviruses, which raises questions about the functions of the common leader sequence and nidovirus transcriptase proteins and the evolution of nidovirus transcription. In this review, nidovirus transcription mechanisms are compared, the experimental systems used are critically assessed and, in particular, the impact of recently developed reverse genetic systems is discussed. The increasing complexity of the nidovirus groupNidoviruses are a group of enveloped positive-stranded RNA viruses. Currently known representatives mostly infect mammals (coronaviruses, toroviruses and arteriviruses), but do also have avian (coronaviruses) or invertebrate (roniviruses) hosts. Nidoviruses cause a variety of diseases, the outcome of which can range from an asymptomatic, persistent carrier-state to a sometimes fatal infection. The severity of coronavirus infection is exemplified by severe acute respiratory syndrome (SARS) in humans, which was caused by a newly emerged coronavirus that gripped worldwide attention in (Drosten et al., 2003Ksiazek et al., 2003;Peiris et al., 2003). In the wake of the SARS outbreak, several other novel coronaviruses, including two that infect humans (van der Hoek et al., 2004; Fouchier et al., 2004;Woo et al., 2005), were identified and added to the growing list of nidoviruses that were first characterized during the past two decades.During that same period of time, the systematic sequence analysis of virus genomes has changed the face of virus taxonomy. With the rise of virus genetics and molecular virology, it has become clear that comparative sequence analysis will provide the most solid basis for future systems for virus classifi...

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona-and toroviruses. The gene contains two large ORFs, ORFla and ORFlb, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORFlb is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORFlb product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORFlb products. The sequences of the EAV and coronavirus ORFla proteins were found to be much more diverged. The EAV ORFla product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

Positive‐stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non‐contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific ‘polymerase jumping’ event.

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3.3 logs (95% CI 2.6-4.0 logs; p<0.001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0.013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5 sequence, the so-called leader RNA, fused at specific positions to different 3 regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3 end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.T ranscriptional regulation is one of the key processes in the controlled expression of genetic information. In general, the transcription of eukaryotic DNA genomes is regulated in the nucleus, where it is subject to pre-, co-, and posttranscriptional control mechanisms (1, 2). In contrast, the transcription of eukaryotic positive-strand RNA [(ϩ)RNA] virus genomes occurs in the cytoplasm and relies on the process of RNAdependent RNA synthesis. In some cases, the genome is the only viral mRNA and gene expression is regulated solely at the (post-)translational level, primarily by the synthesis and controlled processing of precursor polyproteins. Alternatively, regulation may occur at the transcriptional level, either by segmentation of the genome or by the generation of one or multiple subgenomic (sg) mRNAs. The latter strategy usually involves the recognition of internal promoter sequences by the viral RNAdependent RNA polymerase (RdRp) complex, a process that resembles the recognition of DNA promoters by DNAdependent RNA polymerases (3, 4).Nidoviruses (arteriviruses and coronaviruses) are mammalian (ϩ)RNA viruses that appear to have evolved the use of sg mRNAs to regulate the expression of their polycistronic genome (5, 6). The nidovirus replicase is expressed from the genomic RNA as a polyprotein, but the structural proteins are translated from a set of six to eight sg mRNAs (Fig. 1). A key feature of these sg transcripts is the fact that their 5Ј and 3Ј terminal sequences are identical to those of the genome. This nested set structure results from a fusion of the ...