Leader-mRNA junction sequences are unique for each subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection

The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNA, 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.