title: Expression of the barley stripe mosaic virus RNA beta "triple gene block".
abstract: Genomic RNA beta of barley strip mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (beta a) and a "triple gene block" consisting of the beta b, beta c, and beta d ORFs that overlap one another. Two subgenomic beta RNAs (sgRNA beta 1 and sgRNA beta 2) with sizes of 2.5 and 0.96 kb were identified in BSMV-infected protoplasts, and their transcription initiation sites were mapped to nucleotides 789 and 2327, respectively, of RNA beta by primer extension experiments. In a cell-free wheat germ translation system, genomic RNA beta served as a mRNA only for the 22-kDa coat protein, and sgRNA beta 1 directed synthesis of only the 58-kDA beta b protein. However, with sgRNA beta 2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14- and the 23-kDa proteins were recognized by the beta d antibodies in vitro and in vivo. These results demonstrated that the 14-kDa protein was encoded by the beta d ORF and suggested that the 23-kDa protein, designated beta d', is a readthrough product of the amber stop codon of the beta d ORF. Mutagenesis of sgRNA beta 2 revealed that the 17-kDa protein was a product of the beta c ORF. Expression of sgRNA beta 1 and sgRNA beta 2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs alpha and gamma plus chimeric RNA beta derivatives containing the CAT gene in-frame with the beta b, beta c, beta d, or beta d' ORFs. Elimination of the sgRNA beta 1 promoter abolished CAT expression from the beta b-CAT chimeric RNA, and removal of the sgRNA beta 2 promoter prevented CAT expression from the beta c-CAT, beta d-CAT, and beta d'-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNA beta, whereas sgRNA beta 1 serves as a messenger for translation of the beta b protein, and sgRNA beta 2 functions as a messenger for translation of beta c and beta d and the newly discovered beta d' protein. Additional mutagenesis experiments indicate that beta c is translated by a leaky scanning mechanism.
authors.affiliation: Department of Plant Biology, University of California at Berkeley, CA 94720-3102, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhou
authors.forename: A O
authors.initials: AO
authors.lastname: Jackson
mesh_terms: D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002874:Chromosome Mapping; D018388:Codon, Terminator; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005807:Genes, Overlapping; D017930:Genes, Reporter; D016679:Genome, Viral; D001467:Hordeum / Q000821:virology; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics*; D016366:Open Reading Frames; D018515:Plant Leaves / Q000821:virology; D014176:Protein Biosynthesis; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D018388:Codon, Terminator; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase