STRUCTURAL ELEMENTS OF 3'-TERMINAL COAT PROTEIN-BINDING SITE IN ALFALFA MOSAIC-VIRUS RNAS

The 3'-termini of the three genomic RNAs of alfalfa mosaic virus (AlMV ) and ilarviruses contain a number of AUGC-motifs separated by hairpin structures. Binding of coat protein (CP) to such elements in the RNAs is required to initiate infection of these viruses, Determinants for CP binding in the 3'-terminal 39 nucleotides (nt) of AlMV RNA 3 were a nalyzed by band-shift assays. From the 5'- to 3'-end this 39 nt sequen ce contains AUGC-motif 3, stem-loop structure 2 (STLP2), AUGC-motif 2, stem-loop structure 1 (STLP1) and AUGC-motif 1, A mutational analysis showed that all three AUGC-motifs were involved in CP binding. Mutati on of the A- and U-residues of motifs 1 or 3 had no effect on CP bindi ng but similar mutations in motif 2 abolished CP binding. A mutational analysis of the stem of STLP1 and STLP2 confirmed the importance of t hese hairpins for CP binding. Randomization of the sequence of the ste ms and loops of STLP1 and STLP2 had no effect on CP binding as long as the secondary structure was maintained. This indicates that the two h airpins are not involved in sequence-specific interactions with CP. Th ey may function in a secondary structure-specific interaction with CP and/or in the assembly of the AUGC-motifs in a configuration required for CP binding.