Regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization

A reporter open reading frame (ORF) coding for a fusion of bacterial beta-g lucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closte rovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BW ORFs encoding the HSP70 homolog (HSP70h), a major capsid p rotein (CP), and a 20-kDa protein (p20) resulted in the expression of the p rocessed GUS-Pro reporter from corresponding subgenomic RNAs. The high sens itivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP pr omoter and later the p20 promoter. The kinetics of transcription of the rem aining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (C Pm), and a 21-kDa protein (p21) were examined via Northern blot analysis. T aken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p6 4 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a d ramatic increase in the level of transcription from one of the two remainin g subgenomic promoters. Comparison with other positive-strand RNA viruses p roducing multiple subgenomic RNAs showed the uniqueness of the pattern of c losterovirus transcriptional regulation.