Second-site mutations in the brome mosaic virus RNA3 intercistronic region partially suppress a defect in coat protein mRNA transcription.

An intercistronic oligo(A) tract is present in the genomic RNA3 of all bromoviruses sequenced to date and, for brome mosaic virus (BMV), is known to function as an activating sequence in transcription of the subgenomic coat protein mRNA, RNA4. Mutations able to partially compensate for removal of the oligo(A) from BMV RNA3 were identified by obtaining spontaneous second-site revertants. The starting BMV RNA3 mutant carried a deletion of 17 of 18 residues of the intercistronic oligo(A), resulting in a nearly complete loss of subgenomic coat protein mRNA synthesis and reduced RNA3 accumulation. The responsible suppressor mutations acted in cis and were located in the 244-base RNA3 intercistronic region containing the original deletion. Three mutations associated with the revertant phenotype were characterized: (i) A single U-->A substitution in the core subgenomic mRNA promoter restored 35% of wild-type promoter activity. (ii) A duplication of 8 bases (UAUUAUUA) immediately 5' to the oligo(A) deletion site resulted in higher levels of both RNA3 and RNA4 accumulation. (iii) A point substitution in a conserved cellular motif corresponding to box B of RNA polymerase III promoters reduced RNA3 accumulation. Together with certain intervirally conserved promoter sequences, the spontaneous adaptation of a bromovirus subgenomic promoter to function without its unusual oligo(A) activator suggests that bromovirus subgenomic mRNA transcription may share underlying mechanistic similarities with the many other members of the alphavirus-like superfamily, whose subgenomic promoters all lack an oligo(A) tract.