CONTRIBUTIONS OF THE BROME MOSAIC-VIRUS RNA-3 3'-NONTRANSLATED REGION TO REPLICATION AND TRANSLATION

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts. Deletion of single stem-loop structures gave progeny retaining near-wild-type accumulation levels. Constructions representing deletion of two or three stem-loops substantially lowered the accumulation of progeny RNA-3 relative to wild-type levels. RNA-3 mutants bearing deletions of longer sequences or of the entire region (DELTAPsKs RNA-3) replicated poorly, yielding no detectable RNA-3 or RNA-4 progeny. Levels of RNA-1 and RNA-2, in the presence of a mutant RNA-3, were found to increase relative to the accumulation observed in a complete wild-type transfection. The stability of DELTAPsKs RNA-3 in protoplasts was somewhat lower than that of wild-type RNA during the first 3 h postinoculation. Little difference in translatability in vitro of wild-type and RNA-3 constructs bearing deletions within the stem-loop region was observed, and Western immunoblot analysis of viral coat protein produced in transfected protoplasts showed that protein accumulation paralleled the amount of RNA4 message produced from the various sequences evaluated. These results indicate that the RNA-3 pseudoknot region plays a minor role in translational control but contributes substantially to the overall replication of the brome mosaic virus genome.