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 (delta PsKs 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 delta PsKs 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 RNA-4 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.
Lahser
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Institute of Developmental and Molecular Biology, Texas A&M University, College Station 77843-3155.
Marsh
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Hall
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D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis; D001467:Hordeum / Q000382:microbiology; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics* / Q000254:growth & development*; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D011523:Protoplasts / Q000382:microbiology; D012313:RNA; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
D012333:RNA, Messenger; D012367:RNA, Viral; C039919:RNA, recombinant; D012313:RNA