MUTATIONS IN COAT PROTEIN-BINDING SITES OF ALFALFA MOSAIC-VIRUS RNA-3AFFECT SUBGENOMIC RNA-4 ACCUMULATION AND ENCAPSIDATION OF VIRAL RNAS

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mos aic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified A MV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding o f 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 in fectious clone of RNA 3, and mutant RNA transcripts were used as templ ates 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-stran d promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Revers ion of three G-C base pairs in hairpin 1 had no effect on CP binding b ut interfered with minus-strand promoter activity in vitro and with RN A 3 replication in vivo. It is concluded that the viral RdRp and CP re cognize different elements in the 3'-UTRs of AMV RNAs. Moreover, sever al mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, bu t not with the accumulation of RNA 3.