Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

Barley yellow dwarf virus RNA lacks both a 5′ cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3′ UTR, that confers efficient translation initiation at the AUG closest to the 5′ end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3′-5′ communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3′ and 5′ UTR contexts. A seven-base loop sequence was needed only in the 3′ UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3′ UTR.