Mutations in Coat Protein Binding Sites of Alfalfa Mosaic Virus RNA 3 Affect Subgenomic RNA 4 Accumulation and Encapsidation of Viral RNAs

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mosaic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified AMV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding of CP to the viral RNAs is required to initiate infection. The sequence of the 3'-terminal 39 nucleotides of AMV RNA 3 can be folded into two stem-loop structures flanked by three single-stranded AUGC sequences and represents a CP binding site. Mutations in this sequence that are known to interfere with CP binding in vitro were introduced into an infectious clone of RNA 3, and mutant RNA transcripts were used as templates in the in vitro RdRp assay and to infect protoplasts and plants. Mutation of AUGC motif 2 or disruption of the stem of the 3'-proximal hairpin 1 interfered with CP binding in vitro but not with minus-strand promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Reversion of three G-C base pairs in hairpin 1 had no effect on CP binding but interfered with minus-strand promoter activity in vitro and with RNA 3 replication in vivo. It is concluded that the viral RdRp and CP recognize different elements in the 3'-UTRs of AMV RNAs. Moreover, several mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, but not with the accumulation of RNA 3.