Site-directed mutagenesis of potential protein-coding regions in expressible cloned cDNAs of cucumber mosaic viral satellites

Abstract

Site-directed mutagenesis was used to alter potential protein-coding regions (open reading frames or ORFs) within the cloned, expressible cDNAs of two satellites of cucumber mosaic virus (CMV), S-CARNA 5 and D-CARNA 5. RNA transcripts synthesized in vitro from the mutant and wild-type satellite cDNAs were tested for biological activity on tomato plants by coinfection with CMV RNAs 1, 2, and 3, and progeny CARNA 5s generated from such transcripts were isolated and sequenced. Two mutants of S-CARNA 5 were constructed in attempts to test whether the ORF (ORF IIB, beginning at nucleotide 135) responsible for synthesis in vitro of two small proteins (M. J. Avila-Rincon, C. W. Collmer, and J. M. Kaper (1986). Virology 152, 455-458) could be eliminated without loss of the satellite's biological activity. Biological tests with mutant transcripts that either lacked the AUG initiation codon or contained a premature translation termination codon were foiled by instability and/or reversion in the progeny. With progeny CARNA 5 of the former mutant, one of two altered nucleotides reverted, thus restoring the AUG but not the nucleotide immediately preceding it. In contrast, a mutation in the necrogenic D-CARNA 5 which altered the initiation codon of ORF I (nucleotides 11-94), whose predicted amino acid sequence is conserved in all necrogenic CMV satellites sequenced to date, was stable in biological testing and did not destroy necrogenicity. This was shown by nucleotide sequencing of the progeny CARNA 5 from necrotic test plants and by its direct comparison with wild-type D-CARNA 5 progeny in a tomato necrosis dilution assay. This experiment provides convincing evidence that a possible protein product of ORF I is not involved in the induction of tomato necrosis by D-CARNA 5.