Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication.

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Selective replication of coronavirus genomes that express nucleocapsid protein.

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.

Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo.

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 ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected 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.

Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA.

A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-alpha displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.

Multigene RNA vector based on coronavirus transcription.

Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5' and 3' ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.

Mechanisms and enzymes involved in SARS coronavirus genome expression.

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 post-translational 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.

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus.

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 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 reverse-genetic 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.