Second-site suppressor mutations assist in studying the function of the 3' noncoding region of turnip yellow mosaiac virus RNA

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57 leads to U mutation in the tRNA-like structure was a strong suppressor of the C-55 leads to A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107 leads to C mutation was a strong second-site suppressor of the U-96 leads to G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G+92 leads to A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A+92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A+92/G-96 double mutant in plants.