Precise mapping and in vitro translation of a trifunctional subgenomic RNA of barley yellow dwarf virus

Abstract

The barley yellow dwarf virus genome consists of a single 5.7-kb RNA encoding six open reading frames (ORFs). The four ORFs in the 3′ half of the genome were proposed to be expressed via subgenomic mRNAs. Here, we show that the PAV serotype of barley yellow dwarf virus generates two subgenomic RNAs of 2.9 and 0.8 kb in infected plants and protoplasts. The 5′ end of the larger subgenomic RNA (sgRNAi) was precisely mapped to base 2769 by ribonuclease protection and primer extension methods. Synthetic sgRNAi, containing the exact 5′ and 3′ ends, was generated by in vitro transcription, translated in vitro, and shown to serve as a message for three ORFs. Translation initiated at the first two AUG codons in sgRNA1, yielding the 22-kDa viral coat protein (CP) and a 17-kDa protein encoded by an overlapping, out-of-frame ORF. In addition, readthrough of the amber stop codon of the CP ORF gave rise to a 72-kDa fusion product of the CP ORF and an in-frame 50K ORF immediately 3′ of the CP termination codon. The 0.8-kb sgRNA2 was present in greater abundance than sgRNAi and likely serves as a message for a 6.7K ORF at the 3' end of the genome. Sequences that may control subgenomic RNA synthesis and the translational events are compared with those of other plant viruses.