The conserved 5′ apical hairpin stem loops of bamboo mosaic virus and its satellite RNA contribute to replication competence

Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5′ apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C(60) and C(83)) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5′ ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.