A CIS-ACTING FUNCTION FOR THE CORONAVIRUS LEADER IN DEFECTIVE INTERFERING RNA REPLICATION

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomi c mRNAs suffices as a 5'-terminal cis-acting signal for RNA replicatio n, a corollary to the notion that coronavirus mRNAs behave as replicon s, synthetic RNA transcripts of a cloned, reporter-containing N mRNA ( mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(li) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but syn thetic transcripts of a cloned reporter-containing defective interferi ng (DI) RNA differing from the N mRNA construct by 433 nt of continuou s 5'-proximal genomic sequence between the leader and the N open readi ng frame did replicate and become packaged, indicating the insufficien cy of the leader atone as a 5' signal for replication of transfected R NA molecules. The leader was shown to be a necessary part of the cis-a cting signal for DI RNA replication, however, since removal of termina l bases that destroyed a predicted intraleader stem-loop also destroye d replicating ability. Surprisingly, when the same stem-loop was disru pted by base substitutions, replication appeared only minimally impair ed and the leader was found to have rapidly reverted to wild type duri ng DI RNA replication, a phenomenon reminiscent of high-frequency lead er switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled fo r initiation of DI RNA replication, the wild-type leader is strongly p referred for subsequent replication. They also demonstrate that, in co ntrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements orig inating from discontinuous downstream regions within the polymerase ge ne for replication or for packaging.