A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and posttranslational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries. INTRODUCTIONSevere acute respiratory syndrome (SARS) is a lifethreatening form of pneumonia (Peiris et al., 2003a). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal-oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b;Drosten et al., 2003;Ksiazek et al., 2003;Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29?7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003;Marra et al., 2003;Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001;Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the 21 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et ...

We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5 and 3 ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.Coronaviruses are enveloped positive-strand RNA viruses with a genome size of approximately 30 kb. More than twothirds of the genome encodes an RNA-dependent RNA-replicase, which is expressed from the viral genomic RNA (13, 21). The replicase gene is comprised of two large overlapping open reading frames (ORFs), ORF1a and ORF1b, which are translated as two polyprotein precursors, pp1a and pp1ab. The larger protein, pp1ab, is expressed by programmed (Ϫ1) ribosomal frameshifting. Extensive processing of the polyproteins by virus-encoded proteinases leads to the formation of a replication-transcription complex in the cytoplasm of the infected cell (31).A key feature of coronaviruses is their unique transcription strategy. This strategy leads to the synthesis of a nested set of 3Ј coterminal subgenomic mRNAs, encoding mainly structural proteins. It has been shown that the synthesis of each subgenomic mRNA involves a discontinuous step by which the socalled 3Ј body sequence is fused to the genomic 5Ј leader sequence (22). This process most probably occurs during the synthesis of subgenomic, negative-strand templates (19,28). The fusion of leader and body sequences during discontinuous transcription is determined, at least in part, by cis-acting elements, termed transcription-associated sequences (TAS). These elements are located at the 5Ј end of the genome and at 3Ј proximal sites corresponding to the individual transcription units (5).Until recently, the study of coronavirus transcription was essentially restricted to the analysis of defective RNA templates that depend upon transcriptional functions provided by a helper virus (15). Nevertheless, some general characteristics of coronavirus transcription have been revealed. Thus, it has been shown that coronavirus-specific transcripts can be generated from defective RNA templates that contain one or several TAS elements (15, 29). Also, mutagenesis of TAS elements in defective RNAs has revealed that TAS base pairing plays an important role in coronavirus discontinuous transcription (27). However, the use of defective RNAs to study coronavirus transcription has focused attention on the template RNA rather than the viral gene products that provide transcriptional functions.Our limited knowledge of coronavirus replicase proteins has been obtained mai...

The coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in coronavirus replication and transcription. We found that human coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one coronavirus structural protein, the N protein, is involved in coronavirus replication.Coronaviruses are enveloped, positive-strand RNA viruses that are mainly associated with enteric or respiratory diseases in humans, companion animals, and livestock. Coronavirus particles contain a genomic RNA of approximately 27,000 to 30,000 nucleotides and four structural proteins: the spike glycoprotein S, the membrane protein M, the small envelope protein E, and the nucleocapsid protein N (44). Three of these four proteins are embedded in the viral envelope. These are the S protein, which mediates binding of the virus particle to the target cell and the subsequent fusion of viral and cellular membranes (8), the M protein, which has a crucial role in the incorporation of the virus nucleocapsid into virus particles (28,30,31), and the E protein, which facilitates virus assembly, possibly by inducing curvature into pre-Golgi membranes, the site at which coronaviruses assemble by budding (13, 36). The fourth structural protein, N, is associated with the viral RNA genome to form a ribonucleoprotein complex (38).The coronavirus N protein has been described as a multifunctional protein displaying RNA-binding activity, proteinprotein interaction (specifically with the M protein), and the ability for self-association (22,29,30). Clearly, many of these features will reflect the structural role of the N protein in the virus particle. However, there are also some observations that suggest the N protein may have a role in viral RNA synthesis. First, several studies have provided evidence for the binding of N protein to coronaviral RNA sequences that are involved in the regulation of RNA synthesis. These sequences include the coronavirus leader sequence, transcription regulatory sequences (TRS), and sequences corresponding to the 3Ј end of coronavirus genomes (2,10,11,24,34,48). Second, it has been shown that in addition to a cytoplasmic distribution within the host cell, at least a fraction of the coronavirus N protein colocalizes with replicative proteins at the site...

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture (ϳ10 9 PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virusinfected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30 000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reversegenetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.