Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify thecis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3′-terminalcis-essential domain of both genomic and DI RNAs can be folded into a stable stem-loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem-loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3′ terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3′-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.
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