A phylogenetically conserved hairpin-type 3 ' untranslated region pseudoknot functions in coronavirus RNA replication

Secondary and tertiary structures in the 3' untranslated region (UTR) of pl us-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a M-nu cleotide (nt) hairpin-type pseudoknot within the 288-nt 3' UTR of the bovin e coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective int erfering RNA genome. The pseudoknot is phylogenetically conserved among cor onaviruses both in location and in shape but only partially in nucleotide s equence, and evolutionary covariation of bases to maintain G U pairings ind icates that it functions in the plus strand. RNase probing of synthetic tra nscripts provided additional evidence of its tertiary structure and also id entified the possible existence of two conformational states. These results indicate that the 3' UTR pseudoknot is involved in coronavirus RNA replica tion and lead us to postulate that it functions as a regulatory control ele ment.