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 segmentspecific 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.Viruses classified as members of the family Reoviridae have been isolated from a wide range of hosts including vertebrates, insects, mollusks, and plants (1). Several members of this virus group are of medical and economic importance; e.g., rotaviruses are a major cause of infant mortality in many parts of the world, and cytoplasmic polyhedrosis viruses are potential insect-control agents (1). Other members provide valuable model systems for molecular, pathogenicity, and epidemiologic studies (1). The genomes of these viruses consist of 10-12 segments of double-stranded RNA (1). Available evidence suggests that each virus particle contains one copy of each segment comprising the genome (2-5). The mechanism responsible for the selective sorting of individual genome segments remains an intriguing unsolved problem presumably involving complex protein-RNA and RNA-RNA interactions. Characterization of functional remnants of genome RNAs generated by deletion events has provided insights into the mechanism of genome replication and encapsidation for members of other virus families (6-9). Although there exists little information regarding remnant RNAs associated with the genome of mammalian reoviruses, there have been several reports describing remnant RNAs associated with isolates of wound tumor virus (WTV), the type member of the genus Phytoreovirus (10-12).WTV replicates both in plant hosts and in several species of leafhoppers, which act as vectors. Maintenance of WTV exclusively in vegetatively propagated plant hosts results in virus populations that are defective in the abil...

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