Viral Replicase Gene Products Suffice for Coronavirus Discontinuous Transcription

Abstract

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...

Synthesis, Crystal Structure, Structure−Activity Relationships, and Antiviral Activity of a Potent SARS Coronavirus 3CL Protease Inhibitor

“…[7][8][9] It consists of about 29 700 nucleotides with its replicase gene alone encompassing more than 21 000 nucleotides and encoding two overlapping polyproteins, pp1a (486 kDa) and pplab (790 kDa). 10 The functional polypeptides are released from each polyprotein through extensive proteolytic processing, primarily by the 34.6 kDa protease, which is also called the 3C-like protease (3CL pro ). Designing small molecule inhibitors to the 3CL protease is an attractive strategy for the development of anti-SARS drugs.…”

Coronavirus Main Proteinase (3CL pro ) Structure: Basis for Design of Anti-SARS Drugs

“…Coronaviruses are positive-stranded RNA viruses featuring the largest viral RNA genomes known to date (27 to 31 kb). The gene for the human coronavirus 229E replicase, encompassing more than 20,000 nucleotides, encodes two overlapping polyproteins [pp1a (replicase 1a, ϳ450 kD) and pp1ab (replicase 1ab, ϳ750 kD) (5)] that mediate all the functions required for viral replication and transcription (6). Expression of the C-proximal portion of pp1ab requires (-1) ribosomal frameshifting (5).…”

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

“…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).…”

“…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).…”