In vivo expression and mutational analysis of the barley yellow dwarf virus readthrough gene

The barley yellow dwarf virus (BYDV) coat protein gene is separated from an adjacent downstream open reading frame (ORF) by a single termination codon. Immunological analysis of this downstream "readthrough" region reveals multiple coat protein-readthrough products. A full-length 72-kDa (P72) coat protein-readthrough fusion product is detected in total lysates from infected cells. However, purified aphid transmissible virions contain only a 50-kDa (P50) coat protein-readthrough product. Virion-associated P50 lacks the C-terminal domain predicted by its ORF sequence. A separate 33-kDa polypeptide (P33) corresponding to the readthrough C-terminus domain is detected in the crude cellular membrane fraction. Site-directed and deletion mutational analysis demonstrate that the readthrough ORF is dispensable for BYDV replication and virion accumulation in protoplasts. In contrast, a mutant which results in a continuous fusion product of coat and readthrough sequences is not viable. Point mutations were used to map regions required for P50 and P72 synthesis. A model explaining the relationships between the three forms of the readthrough polypeptides is proposed.