Cloning and sequence analysis of a cDNA encoding the capsid protein of the MAV isolate of barley yellow dwarf virus.

We have generated a eDNA clone of the MAY isolate of barley yellow dwarf virus (BYDY) containing the entire capsid protein coding region. The eDNA was synthesized by the method of Gubler and Hoffman (1) using an oligonucleotide primer (5'-GTCTACCTATTTGG-3') complementary to the 3' terminus of the capsid protein gene of the PAY isolate of BYDY (2). A pUC18 derivative (pMAY1) containing a 3.5 eDNA insert was identified by restriction enzyme analysis. Nucleotide sequence analysis of both ends of this eDNA insert and comparison to the corresponding segments of the PAY-BYDY genome indicated that the insert is co-linear with genomic viral RNA and contains the capsid protein gene. A 0.66 kb eDNA fragment from pMAY1 containing the MAY-BYDY capsid protein coding region plus a 55 nt upstream noncoding leader sequence was inserted into transcription vector pBS M 13 + in the plus-sense direction downstream of the bacteriophage T7 promoter. Resultant in vitro-generated transcripts were translated in a rabbit reticulocyte lysate to yield a major protein product that comigrated with authentic MAY-BYDY virion capsid protein and a minor 17000 Mr protein product. A nested set of deletions covering both eDNA strands of the MAY-BYDY capsid protein gene was constructed using exonuclease III (3) and was subsequently sequenced by the dideoxy chain termination method (4). The ORF of the capsid protein gene (Fig. 1) consists of 600 nt (including the stop codon) and encodes a 21848 Mr protein; the levels of homology between the capsid protein ORF's of MAY-BYDY and PAY-BYDY are 76.5% at the nucleotide level and 72.6% at the protein level. In addition, a second ORF internal to but in a different reading frame than the capsid protein gene ORF exists between nucleotides 38 and 502; this ORF may encode a 17206 Mr protein.Figure 1. Nucleotide sequence of the MA V-BYDV capsid protein gene and the deduced amino acid sequence. The initiation and termination codons are underlined.