Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo.

DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the 5'-proximal P3 gene, encoding the putative viral transport function, did not affect replication whereas deletions in the 3'-proximal coat protein gene reduced replication of RNA 3 by about 100-fold. Sequences required for the synthesis in protoplasts of RNA 4, the coat protein messenger, were more extensive than the subgenomic promoter characterized previously in an in vitro replicase assay. At the 5'-end of RNA 3 a sequence of 169 nucleotides was sufficient for replication whereas a sequence of 112 nucleotides was not. 3'-Terminal deletions up to 133 nucleotides reduced replication to a low but significant level. Further 3'-deletions abolished replication.