Expression of the barley stripe mosaic virus RNAβ "triple gene block"

Abstract

Genomic RNAβ of barley stripe mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (βa) and a "triple gene block" consisting of the βb, βc, and βd ORFs that overlap one another. Two subgenomic β RNAs (sgRNAβ1 and sgRNAβ2) 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 RNAβ by primer extension experiments. In a cell-free wheat germ translation system, genomic RNAβ served as a mRNA only for the 22-kDa coat protein, and sgRNAβ1 directed synthesis of only the 58-kDa βb protein. However, with sgRNAβ2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14-and the 23-kDa proteins were recognized by the βd antibodies in vitro and in vivo. These results demonstrated that the 14-kDa protein was encoded by the βd ORF and suggested that the 23-kDa protein, designated βd′, is a readthrough product of the amber stop codon of the βd ORF. Mutagenesis of sgRNAβ2 revealed that the 17-kDa protein was a product of the βc ORF. Expression of sgRNAβ1 and sgRNAβ2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs α and γ plus chimeric RNAβ derivatives containing the CAT gene in-frame with the βb, βc, βd, or βd′ ORFs. Elimination of the sgRNAβ1 promoter abolished CAT expression from the βb-CAT chimeric RNA, and removal of the sgRNAβ2 promoter prevented CAT expression from the βc-CAT, βd-CAT, and βd′-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNAβ, whereas sgRNAβ1 serves as a messenger for translation of the βb protein, and sgRNAβ2 functions as a messenger for translation of βc and βd and the newly discovered βd′ protein. Additional mutagenesis experiments indicate that βc is translated by a leaky scanning mechanism. © 1995 Academic Press, Inc.