Structural Elements of the 3'-Terminal Coat Protein binding Site in Alfalfa Mosaic Virus RNAs

Abstract

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 analyzed by band-shift assays. From the 5′-to 3′-end this 39 nt sequence 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. Mutation of the A-and U-residues of motifs 1 or 3 had no effect on CP binding but similar mutations in motif 2 abolished CP binding. A mutational analysis of the stem of STLP1 and STLP2 confirmed the importance of these hairpins for CP binding. Randomization of the sequence of the stems 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 hairpins are not involved in sequence-specific interactions with CP. They 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.

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

“…3C, Table 1) strongly suggests that the hairpin is essential for forming the RNA-coat protein complex. This result is consistent with mutagenesis data showing that disrupting the hairpin was accompanied by a loss of coat protein binding, while restoring the hairpin with different sequences was permissive (15,26). An unexpected finding was that G-U wobble pairs in hairpin 847 to 864 support high-affinity coat protein binding (Fig.…”

“…4I and J), leading to 50-and 15-fold decreases in coat protein binding affinities, respectively. Reusken and Bol reported previously that converting the CϭG pairs at 869/877 and 870/876 to GϭC pairs retained coat protein binding potential (26). Coupled with the strong nucleotide conservation in the selection, these results suggest that the stability of C-G base pairs in hairpin 869 to 877 is an important determinant of AMV coat protein binding.…”

“…Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28). An amino acid mutation that disrupts coat protein binding to the viral RNA (R17A) also blocks RNA replication (37), suggesting that coat protein binding is directly linked to replication functions.…”

The 3' untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element.

“…2 (b) shows that RNAs with a mutation in AUGC motif 1, 2, 3, 4 or 5 (lanes 3-7) or a mutation in motifs 3 and 5 (lane 8) had template activity similar to that of the wt transcript (lane 2). Previously, we showed that mutations in motifs 2 and 3 affected CP binding to site I in the 3h UTR of RNA3 in vitro whereas mutations in motifs 4 and 5 affected CP binding to site II (Reusken et al, 1994 ;Reusken & Bol, 1996). In addition, we observed that replacing AUGC motif 2 or 3 by AGGC reduced or abolished accumulation of RNA3 in plants, respectively (van der Vossen et al, 1994).…”

“…A CP binding site in the 3h-terminal 39 nt of RNA3 has been analysed in most detail (Houser-Scott et al, 1994Reusken & Bol, 1996). Binding of CP to the three genomic AMV RNAs is required to initiate infection (Bol et al, 1971 ;Smit et al, 1981).…”