Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3

During infection of Tetragonia expansa leaves, RNA 3 of the quadripartite genome of beet necrotic yellow vein virus directs synthesis of a subgenomic RNA (RNA 3sub) which corresponds to the 3'-terminal 600 residues of the RNA 3 molecule. Biologically active run-off transcripts have been prepared from full-length cDNA of RNA 3 cloned behind a bacteriophage T7-RNA polymerase promoter. RNA 3 transcripts carrying deletions in the vicinity of the RNA 3sub initiation site were produced by site-directed mutagenesis at the cDNA level and then tested for their capacity to direct RNA 3sub synthesis in infected leaves. The cis-acting domain essential for normal levels of RNA 3sub production in planta (the 'core' promoter) did not extend in the 5'-direction beyond position -16 relative to the RNA 3sub transcription initiation site. The 3'-boundary of the core promoter domain was located somewhere between positions +100 and +208. Displacement of the promoter domain to an upstream site in RNA 3 produced a new subgenomic RNA starting at or near the predicted upstream site.