Recombination and polymerase error facilitate restoration of infectivity in brome mosaic virus

The tRNA-like structure present in the 3' noncoding region of each of the four virion RNAs of brome mosaic virus possesses a conserved A-67-U-A-65 (67AUA65) sequence. Four mutations in this region (67UAA65, 67GAA65, and 67CAA65, each with a double base change, and 67GUA65, containing a single point mutation), previously shown in vitro to be defective in minus-strand promoter function, were introduced into full-length genomic RNAs 2 and 3, and their replicative competence was analyzed in barley protoplasts. All four RNA 3 mutants were capable of replication, although progeny plus-sense RNA 3 accumulation was only 12 to 42% of that of the wild type. Replication of RNA 2 transcripts bearing these mutations was even more severely debilitated; the accumulation of each mutant progeny plus-strand RNA 2 was < 10% of that of the wild type. Analysis of mutant RNA 3 progeny recovered from local lesions induced in Chenopodium hybridum and systemic infections in barley (Hordeum vulgare) plants revealed that the mutant base at position 67 from the 3' end had in each case been modified to an A. These changes generated RNAs with functional pseudorevertant (67AAA65 for mutants 67UAA65, 67GAA65, and 67CAA65) or revertant (67GUA65-->67AUA65) sequences. In most instances, the presence of internal markers permitted discrimination between polymerase error and RNA recombination as the process by which sequence restoration occurred. The pseudorevertant sequence was found to be capable of persistence during subsequent propagation in plants when present on RNA 3 but not when present on RNA 2. These data document the fluidity of the RNA genome and reveal situations in which polymerase error or recombination can function preferentially to restore an optimal sequence. They also support the concept that RNA viruses frequently exist as quasispecies and have implications concerning evolutionary strategies for positive-strand RNA viruses.