EXPRESSION OF THE BARLEY STRIPE MOSAIC-VIRUS RNA-BETA TRIPLE GENE BLOCK

Genomic RNA beta of barley stripe mosaic virus (BSMV) contains four de fined 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 site s were mapped to nucleotides 789 and 2327, respectively, of RNA beta b y primer extension experiments. In a cell-free wheat germ translation system, genomic RNA beta served as a mRNA only for the 22-kDa coat pro tein, and sgRNA beta 1 directed synthesis of only the 58-kDa beta b pr otein. 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 we re recognized by the beta d antibodies in vitro and in vivo. These res ults demonstrated that the 14-kDa protein was encoded by the beta d OR F and suggested that the 23-kDa protein, designated beta d', is a read through 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 sgRNAP2 was also investiga ted with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs alpha and gamma plus chimeric RNA be ta 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 a bolished CAT expression from the Pb-CAT chimeric RNA, and removal of t he sgRNAP2 promoter prevented CAT expression from the beta c-CAT, beta d-CAT, and beta d'-CAT chimeric RNAs. Taken together, these results d emonstrate that the BSMV coat protein is the sole translation product of the genomic RNA beta, whereas sgRNA beta 1 serves as a messenger fo r translation of the beta b protein, and sgRNA beta 2 functions as a m essenger for translation of beta c and beta d and the newly discovered beta d' protein. Additional mutagenesis experiments indicate that bet a c is translated by a leaky scanning mechanism. (C) 1996 Academic Pre ss, Inc.