cis-acting elements involved in replication of alfalfa mosaic virus RNAs in vitro

A DNA copy of alfalfa mosaic virus (AIMV) RNA3 was transcribed in vitro in two different orientations with T7 RNA polymerase and the transcripts were used as templates for a virus-specific RNA-dependent RNA polymerase (RdRp) purified from AIMV-infected bean plants. Minus-stranded templates were transcribed by the RdRp into subgenomic plus-stranded RNA4. A deletion analysis showed that a sequence in minus-strand RNA3, located between nucleotides -8 and -55 upstream of the initiation site for RNA4 synthesis, was sufficient for subgenomic promoter activity in vitro. Plus-stranded templates were transcribed by the RdRp into full-length minus-stranded copies. A deletion analysis indicated that a sequence located between nucleotides 133 and 163 from the 3'-end of AIMV RNA3 was sufficient to direct the synthesis of minus-stranded products by the RdRp. Thus, the 3'-terminal region of the AIMV RNAs, which contains the binding sites with a high affinity for coat protein, appears not to be involved in recognition of the RNAs by the RdRp in vitro.