Nucleotides into Open Reading Frame 1

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-replicatio