Characterization of an essential RNA secondary structure in the 3 ' untranslated region of the murine coronavirus genome

We have previously identified a functionally essential bulged stem-loop in the 3' untranslated region of the positive-stranded RNA genome of mouse hep atitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequenc e, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis vir us defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirm ed most of the previously proposed structure, but they revealed that the te rminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Furt her, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and par tially overlapping, RNA pseudoknot that has recently been shown to be neces sary for bovine coronavirus replication.