Comparison of the role of 5' terminal sequences of alfalfa mosaic virus RNAs 1, 2, and 3 in viral RNA replication.

The 5' untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5' Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5' UTR. When the 5' UTR of RNA 3 was replaced by the 5' UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5' UTR of this RNA was replaced by 5' terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5' 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5' 79 nt. However, when the 5' 22 nucleotides of RNA 3 were replaced by the complete 5' UTR of RNA 1 or 5' sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5' viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80-345 of the 5' UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5' end of the RNA.