LOCALIZATION OF CIS-ACTING SEQUENCES ESSENTIAL FOR CYMBIDIUM RINGSPOTTOMBUSVIRUS DEFECTIVE INTERFERING RNA REPLICATION

The smallest defective interfering RNA (DI-2) of cymbidium ringspot to mbusvirus (CyRSV) was used to identify the cis-acting sequences necess ary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleot ides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specif ically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.