Replication of barley yellow dwarf virus RNA and transcriptional control of gene expression

Abstract

Barley yellow dwarf luteovirus (BYDV) generates three 3'-cotenninal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNAl is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNAl transcription start site was mapped previously to either nucleotides 2670 or 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the SgRNAl promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SLl and SL2) in the sgRNAl promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary stmcture, but not the nucleotide sequence was important at the base of SLl. Regions with both RNA primary and secondary structural features that contributed to