The 288-nucleotide (nt) 3' untranslated region (UTR) in the genome of the bovine coronavirus (BCoV) and 339-nt 3' UTR in the severe acute respiratory syndrome (SARS) coronavirus (SCoV) can each replace the 301-nt 3' UTR in the mouse hepatitis coronavirus (MHV) for virus replication, thus demonstrating common 3' cis-replication signals. Here, we show that replacing the 209-nt MHV 5' UTR with the ∼63%-sequence-identical 210-nt BCoV 5' UTR by reverse genetics does not yield viable virus, suggesting 5' end signals are more stringent or possibly are not strictly 5' UTR confined. To identify potential smaller, 5'-common signals, each of three stem-loop (SL) signaling domains and one inter-stem-loop domain from the BCoV 5' UTR was tested by replacing its counterpart in the MHV genome. The SLI/II domain (nucleotides 1 to 84) and SLIII domain (nucleotides 85 to 141) each immediately enabled near-wild-type (wt) MHV-like progeny, thus behaving similarly to comparable 5'-proximal regions of the SCoV 5' UTR as shown by others. The inter-stem-loop domain (nt 142 to 173 between SLs III and IV) enabled small plaques only after genetic adaptation. The SLIV domain (nt 174 to 210) required a 16-nt extension into BCoV open reading frame 1 (ORF1) for apparent stabilization of a longer BCoV SLIV (nt 174 to 226) and optimal virus replication. Surprisingly, pleiomorphic SLIV structures, including a terminal loop deletion, were found among debilitated progeny from intra-SLIV chimeras. The results show the inter-stem-loop domain to be a potential novel species-specific cis-replication element and that cis-acting SLIV in the viral genome extends into ORF1 in a manner that stabilizes its lower stem and is thus not 5' UTR confined.
Available at: http://works.bepress.com/david_brian/2/