Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5 and 3 coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (؊17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5 end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.Coronaviruses and arteriviruses, both members of the Nidovirus order of plus-strand RNA animal viruses, appear unique among RNA viruses in their use of a discontinuous transcription step during synthesis of subgenomic mRNAs (10,14,28,48,54). In both groups of viruses, the transcription pathway ultimately yields a 3Ј coterminal nested set of subgenomic mRNAs that are also 5Ј coterminal with the virus genome. The common 5Ј-terminal sequence, called the "leader," encoded at the genome 5Ј terminus, makes up only a portion of the 5Ј untranslated region in the genome and in each subgenomic mRNA (sgmRNA) species. In general, translation occurs most abundantly from the 5Ј-most open reading frame (ORF) on each sgmRNA. When originally described, the leader was postulated to become fused with the sgmRNA species by a leader-priming mechanism wherein the RdRp undergoes a copy choice jump on the virus genome-length minus-strand template during plus-strand synthesis (4). The jump in this model would occur for each sgmRNA molecule synthesized, and a postulated 3Ј35Ј exonuclease would trim the large primer (80 to 140 nucleotides [nt]), termed free leader, d...
The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3' end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junction region, and in its place was the nonconforming sequence GGTAGAC that occurs just 15 nt downstream in the genome. Leader-mRNA junction sequences found after 296 days of persistent infection were the same as those found during acute infection (< 18 hr postinfection). These data indicate that, in contrast to the closely related mouse hepatitis virus, the bovine coronavirus maintains a stable leader-mRNA junction sequence for each mRNA. Interestingly, this stability may be related to the fact that a UCUAA sequence element, postulated by others to be a regulator of the leader-mRNA fusion event, occurs only once within the 3' flanking sequence of the genomic leader donor and once at intergenic sites in the bovine coronavirus genome, whereas it occurs two to four times at these sites in the mouse hepatitis coronavirus.
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