Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities

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Abstract
A remarkable feature of the genomic RNAs of several plant viruses is the presence at the 3' end of a region that exhibits tRNA-like functions, including aminoacylation. The three genomic and single subgenomic RNAs of brome mosaic virus (BMV) accept tyrosine in vitro and in vivo, the smallest 3' fragment that can be aminoacylated being about 135 nucleotides long. The roles of the tRNA-like properties are incompletely understood, but an involvement in replication rather than translational functions is likely. We have recently shown (J.J.B. et al., in preparation) that the features recognized by the BMV RNA-specific RNA-dependent RNA polymerase (replicase) for the use of BMV RNA for complementary strand synthesis also lie within the tRNA-like structure. To distinguish between the roles of BMV RNA as a substrate for tyrosyl-tRNA synthetase and BMV replicase, we have now produced BMV RNAs with mutations at two separate loci within the tRNA-like structure. This has been achieved by transcription in vitro from specifically mutagenized cDNA, an approach permitting the generation of targeted mutants without regard to their viability in vivo.