Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

Abstract

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.The etiologic agent causing severe acute respiratory syndrome (SARS) is a novel coronavirus (CoV) (8,10,(16)(17)(18)21). This virus causes a life-threatening respiratory disease for which no fully efficacious therapy is available. SARS-CoV is a member of group 2 of the Coronaviridae family within the order Nidovirales (13), which is composed of enveloped, singlestranded, positive-sense RNA viruses relevant in animal and human health (5, 9). Two-thirds of the 29.7-kb SARS-CoV genome carries the replicase gene, which comprises two overlapping open reading frames, ORF 1a and ORF 1b, the latter being translated by a ribosomal frameshift mechanism (29). Translation of both ORFs results in the synthesis of two polyproteins that are processed by viral proteinases to release the components of the replication-transcription complex (36,37). Besides containing RNA-dependent RNA polymerase, RNA helicase, and proteases (4,12,15,23,37), which are all common to positive-strand RNA viruses, the CoV replicase was recently predicted to contain a variety of RNA-processing enzymes that are extremely rare or absent in other RNA viruses, including endoribonuclease (NendoU), 3Ј-to-5Ј exoribonuclease (ExoN), 2Ј-O-ribose methyltransferase (2Ј-O-MT), ADP ribose 1ЈЈ-phosphatase, and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase (25, 36). These enzymatic activities might be involved in the replication of the largest known RNA virus genome and in the production of an extensive set of 5Ј-and 3Ј-coterminal subgenomic RNAs (11,14,25,36).The study of CoV molecular biology has been profoundly advanced by the recent construction of full-length cDNA clones (3,6,26,27,(32)(33)(34) and self-replicating RNAs, or replicons (2,28,30). Due to the large size of the CoV RNA genome and the instability of some CoV replicase gene sequences in bacteria, cDNA clones and replicons have been engineered using bacterial artificial chromosomes (BACs) (3), in vitro ligation of CoV cDNA fragments (32), and vaccinia virus as a vector for the propagation of CoV full-length cDNAs (27). Recently, a SARS-CoV full-length cDNA clone has been generated by the approach of using the in vitro li...

Nidovirus ribonucleases: Structures and functions in viral replication

“…), but no differences in plaque size were seen in these mutants. Similarly, substitutions of an NendoU active-site His using SARS-CoV and HCoV-229E replicon systems 88,89 were tolerated but caused a reduction of viral RNAs when compared to cells carrying the wt replicon RNA (Hertzig T and Ziebuhr J, unpublished).…”

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

“…Indeed, cap methylation genes of many (ϩ)-RNA viruses, such as alphaviruses and flaviviruses, were shown to play a crucial role for efficient virus replication (20,24,(42)(43)(44). In the case of CoVs, a functional and genetic analysis performed on MHV temperature-sensitive mutants mapped to the N7-MTase domain of CoV nsp14 and in the 2ЈO-MTase nsp16 indicated that both are involved in positive strand RNA synthesis by previously formed replicase-transcriptase complexes (13).…”

“…The involvement of nsp14 N7-MTase and of nsp16 2ЈO-MTase in the capping pathway was recently demonstrated biochemically (1,18,20). Moreover, both nsp14 and -16 play crucial roles for efficient RNA synthesis within the SARS-CoV replicon and for transcription/replication of MHV-CoV (13,24).…”

The role of programmed-1 ribosomal frameshifting in coronavirus propagation

“…However recent advances in the stability of plasmid vectors and the availability of class II restriction endonucleases or restriction enzymes that cleave adjacent to the recognition sequence have circumvented some of these difficulties (43). The available clones and replicons (44)(45) have allowed many groups to readily investigate different aspects of the coronavirus lifecycle. Developments in the NMR field are also enabling the solution of larger RNA structures such as those that direct -1 PRF.…”