Barley yellow dwarf luteovirus (BYDV)-PAV serotype, an economically important virus of small grain cereals, has a positive-sense RNA genome encoding at least six open reading frames (ORFs). The goal of the research was to determine the genes and sequences involved in the viral replication and to design efficient antiviral strategies to BYDV-PAV. Genetically engineered resistance is essential for BYDV as the natural resistance is inadequate. Antiviral constructs such as sense RNA, antisense RNA and viral polymerase gene were tested for their ability to reduce virus titre in oat protoplasts as monitored by enzyme-linked immunosorbent assay. All antiviral constructs yielded low levels of viral antigen. However, none of the above constructs showed decrease in viral RNA accumulation in Northern blot analysis. Deletion and mutation analyses were performed to determine genes and cis-acting signals involved in translation, replication and encapsidation of BYDV-PAV. ORFs 1 and 2, which encode the putative polymerase gene, were required for replication. Deletion of the coat protein (CP) gene reduced the accumulation of genomic RNA. The carboxy-terminally extended form of the CP was not necessary for replication or encapsidation. Cis-acting RNA signals in and around ORF6 were essential for viral replication. BYDV-PAV replication may be coupled to translation because defective RNAs containing various deletions were not replicated in trans by the co-inoculated wild-type helper genome. Site-directed mutagenesis was used to map the subgenomic RNA1 (sgRNA1) promoter because subgenomic promoters are putative hotspots of viral recombination and putative replication origin. Mutating the sgRNA1 transcription initiation base, G at 2670, or the nucleotides immediately flanking it, reduced sgRNA1 accumulation. Computer-predicted secondary structures in the putative sgRNA1 promoter regions of many members of subgroup I luteovirus has revealed a conserved stem-loop structure near the sgRNA1 start site. Altering the conserved ACAAA motif reduced both the genomic RNA and sgRNA1 accumulation. A premature stop codon introduced at base 2650, 90 bases from the 3\u27 end of the polymerase gene, abolished BYDV-PAV replication in oat protoplasts
