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Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.