Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 30, 2001
Publication Date: N/A
Interpretive Summary: Barley yellow dwarf viruses (BYDVs) cause the most damaging virus disease of cereal crops. These viruses have a single small chromosome that is expressed and copied (or replicated) by mechanisms that are very different from those used for the chromosomes of their host plants. As such, the features of the BYDV life cycle that are unique to the viruses are potential targets for the control of the BYDVs. Because of the structure of the BYDV chromosomes, it can be difficult to study the action of BYDV genes and/or parts of the chromosome. In this study, we developed a system to study the function of BDYV genes and chromosome segments. We identified sequences required for the replication of chromosomes and the synthesis of sub-chromosomal fragments. This information will be useful to scientists who are interested in studying the mechanisms of replication of BYDVs and devising new methods for virus control.
Technical Abstract: A full-length cDNA of the RNA genome of an Illinois isolate of the PAV strain of Barley yellow dwarf virus (BYDV-PAV-IL) was constructed and placed downstream of the Cauliflower mosaic virus-35S promoter. The biological activities of wild-type and mutant cDNAs were characterized in oat protoplasts. Linear DNA molecules were found to be infectious, expressed the 22-kDa coat protein, and produced subgenomic RNAs (sgRNAs) in ratios similar to those observed in protoplasts inoculated with viral RNA. The transcription start sites of the sgRNAs were determined and found to be similar to those reported for an Australian isolate of BYDV-PAV. However, deletion of sequences distal to the transcription start site of sgRNA2 affected the fidelity of transcription initiation of sgRNA2. Duplication of the genomic regions containing the three sgRNA promoters resulted in the synthesis of additional sgRNAs. Duplication of the sgRNA1 promoter region produced strong negative polar effects on upstream promoters that could be relieved, at least partially, by deleting sequences upstream of the core sgRNA1 promoter. The non-coding region between open reading frame (ORF) 2 and ORF3 was not required for either transcription of sgRNA1 or translation of ORF3, while the non-coding region downstream of ORF6 was required for replication of BYDV-PAV-IL. These results illustrate that in vivo-transcribed cDNA clones can be effective tools for analyzing the expression and replication of the BYDV-PAV genome.