The 3' cis-acting genomic replication element of the severe acute respiratory syndrome coronavirus can function in the murine coronavirus genome

Abstract

The 3' untranslated region (3' UTR) of the genome of the severe acute respiratory syndrome coronavirus can functionally replace its counterpart in the prototype group 2 coronavirus mouse hepatitis virus (MHV). By contrast, the 3' UTRs of representative group 1 or group 3 coronaviruses cannot operate as substitutes for the MHV 3' UTR.

FIG. 1.

Comparison of coronavirus 3′ UTR sequences and structures. Top panel, sequence alignment of the 3′ UTRs of SARS-CoV, MHV, BCoV, TGEV, and IBV. Nucleotides are numbered from the first base at the 3′ end of the genome, excluding poly(A); dashes represent gaps introduced in the alignment. Labeled and underlined features are as follows: (i) stems 1 and 2 of the pseudoknot that is common to the first four sequences and potentially present in the IBV sequence, (ii) the octanucleotide motif (oct) that is conserved in all coronavirus genomes, and (iii) a 32-nt region (IBV motif) found in the IBV and SARS-CoV 3′ UTRs that is identical to segments found near the 3′ ends of the genomes of several astroviruses and an equine rhinovirus (19). The alignment of the MHV sequence (strain A59, accession no. M80644) and BCoV sequence (strain Mebus, accession no. M16620) is as presented previously (6). The SARS-CoV sequence is the Urbani strain (accession no. AY278741) (22); the TGEV sequence is the Purdue strain (accession no. M14878) (7). The IBV 3′ UTR was determined from a clone, pSGIBVUTR, which we generated from an isolate of the Beaudette strain, the 505-nt sequence of which is identical to that found under accession no. AJ311362 (2), except for nt 147. The identity of this variant base in our particular isolate was confirmed from an RT-PCR product obtained from the virus. Bottom panel, the conserved, functionally essential secondary structure found at the upstream ends of the 3′ UTRs of the group 2 coronaviruses MHV and BCoV (4-6, 30) and which is also predicted for the 3′ UTR of SARS-CoV. The structure comprises a bulged stem-loop and a partially overlapping RNA pseudoknot. BCoV nucleotides that differ from those of MHV are circled. Numbering is the same as in the linear sequence alignment, and the N gene stop codon is boxed. Broken lines indicate alternative base pairings for the stem-loop or for pseudoknot stem 1.

FIG. 2.

Replacement of the MHV 3′ UTR by targeted RNA recombination. Interspecies chimeric virus fMHV.v2 (4) contains the ectodomain-encoding region of the feline infectious peritonitis virus (FIPV) S gene (hatched rectangle), which allows it to grow in feline cells but not in murine cells. In addition, in fMHV.v2 the MHV structural protein genes downstream of S are in a rearranged order. MHV recombinants containing the SARS-CoV 3′ UTR (solid rectangle) were generated by transfection of fMHV.v2-infected feline cells with donor RNA transcribed from transcription vector pSGSARUTR. Recombinants were selected as progeny able to grow in murine cells. At the bottom is shown a portion of sequence containing the junction between the MHV N gene and the SARS-CoV 3′ UTR in an RT-PCR product obtained from one particular plaque-purified recombinant, Alb424. For pSGSARUTR-derived RNA, [1] and [HE] indicate fragments from the 5′ end of the MHV genome and the hemagglutinin gene (10); for the fMHV.v2 genome, [M] and [4] indicate fragments of the M gene and gene 4 (4).

FIG. 3.

Phenotype of the SARS-CoV 3′ UTR recombinant. (A) Comparison of plaques of the SARS-CoV 3′ UTR recombinant (Alb424) and the wild type (Alb240). Plaque titrations were done at 37°C on mouse L2 cells; monolayers were stained with neutral red at 48 h postinfection and were photographed 18 h later. (B) Single-step growth kinetics. Confluent monolayers of 17Cl1 cells were infected with Alb424 and Alb240 at a multiplicity of 0.6 PFU per cell. At the indicated times postinfection, aliquots of medium were removed and infectious titers were determined on mouse L2 cells. Open and solid squares (Alb424) and circles (Alb240) represent results from two independent experiments. (C) RNA synthesis. Infected or mock-infected 17Cl1 cells were metabolically labeled with [³³P]orthophosphate in the presence of actinomycin D, and RNA was isolated and electrophoretically analyzed in 1% agarose containing formaldehyde as described previously (17). gRNA, genomic RNA.