HOST EFFECTS AND SEQUENCES ESSENTIAL FOR ACCUMULATION OF DEFECTIVE INTERFERING RNAS OF CUCUMBER NECROSIS AND TOMATO BUSHY STUNT TOMBUSVIRUSES

Passage of cucumber necrosis virus (CNV) containing defective interfer ing (DI) RNAs through cucumber plants decreased the accumulation of Dt RNAs to undetectable levels. Subsequent passages in two Nicotiana spe cies (Nicotiana benthamiana or N. clevelandii) resulted in the appeara nce of DI RNA species that were larger than the DI RNAs observed durin g exclusive serial passages of CNV through the Nicotiana species. Sequ ence analysis of cloned cDNAs corresponding to the two DI RNA populati ons indicated that the smaller CNV-DI RNAs contained the four conserve d regions (I through IV) of the genome typical of tombusvirus DI RNAs, whereas the larger DI RNAs were of similar organization but had a dir ect 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 compara tive 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 [I ind icated that smaller segments of 75 and 35 nucleotides (nt), respective ly, could be deleted without abolishing infectivity. The dispensable s equences in region II of both TBSV and CNV DI RNAs mapped to the top p ortion of a putative stem-loop structure. These studies indicate that both essential and nonessential sequences are conserved in DI RNAs. Th e essential sequences in regions I, II, and IV likely contain importan t cis-acting elements, whereas nonessential regions such as region III may play secondary roles such as optimally spacing cis-acting element s or maintaining the DI RNA at an overall size that is stable. (C) 199 5 Academic Press, Inc.