Passage of cucumber necrosis virus (CNV) containing defective interfering (DI) RNAs through cucumber plants decreased the accumulation of DI RNAs to undetectable levels. Subsequent passages in two Nicotiana species (Nicotiana benthamiana or N. clevelandii) resulted in the appearance of DI RNA species that were larger than the DI RNAs observed during exclusive serial passages of CNV through the Nicotiana species. Sequence analysis of cloned cDNAs corresponding to the two DI RNA populations indicated that the smaller CNV-DI RNAs contained the four conserved regions (I through IV) of the genome typical of tombusvirus DI RNAs, whereas the larger DI RNAs were of similar organization but had a direct repeat of the middle portion of the molecule. This result suggests that the host has an influence on the type of DI RNA that accumulates during consecutive high multiplicity of infection passages. A comparative analysis of deletions targeting the individual conserved regions in both CNV and tomato bushy stunt virus (TBSV) DI RNAs revealed that only region III was completely dispensable for accumulation of either DI RNA species. More refined deletion analyses in regions I and II indicated that smaller segments of 75 and 35 nucleotides (nt), respectively, could be deleted without abolishing infectivity. The dispensable sequences in region II of both TBSV and CNV DI RNAs mapped to the top portion of a putative stem-loop structure. These studies indicate that both essential and nonessential sequences are conserved in DI RNAs The essential sequences in regions I, II, and IV likely contain important cis-acting elements, whereas nonessential regions such as region III may play secondary roles such as optimally spacing cis-acting elements or maintaining the DI RNA at an overall size that is stable.
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