Cofolding organizes alfalfa mosaic virus RNA and coat protein for replication

Abstract

Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3' termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3' conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication.

Fig. 1

(A) Stereoimage of the AMV RNA-CP complex. The RNA is shown in green and the CP26 peptides are shown in gold. (B) Schematic diagram of the AMV RNA and CP26 protein contacts. The AUGC sequences have been color coded. The 5´ AUGC is colored green, the interhelical AUGC sequence is colored purple, and the 3´ AUGC sequence is colored red. Hydrogen-bonding contacts between the RNA and peptide are indicated with filled arrows and van der Waals contacts are indicated by open arrows. #1, peptide 1; #2, peptide 2; G, Gly; K, Lys; P, Pro; Q, Gln; R, Arg; S, Ser; Y, Tyr. (C) Secondary structure of the 39-nucleotide minimal binding domain found at the 3´ end of AMV RNAs, AMV_843-881. The AUGC sequences are color coded as in (B). Base pairs are indicated by brackets.

Fig. 2

Formation of inter-AUGC base pairs. (A) RNA-peptide interactions at the base of duplex 1. The CP peptide a helix and sidechains are shown in gold. The RNA is shown in gray. The 5´ AUGC sequence is colored green, and the interhelical AUGC sequence is colored purple. (B) Similar RNA-peptide interactions are seen at the base of duplex 2. The interhelical AUGC sequence is highlighted in purple and the 3´ AUGC sequence is colored red. R, Arg; Y, Tyr.

Fig. 3

Interactions between the AMV RNA and CP26 peptides. (A) View of the two additional base pairs at the base of duplex 1 and their interaction with peptide sidechains. The carbon atoms of base pairing members at the base of duplex 1 from the 3´ AUGC [U(844) and C(846)] are colored green, whereas members from the interhelical AUGC [A(865) and G(867)] are colored purple. Hydrogen bonds are indicated in cyan. (B) View of peptide 2 making the L-shaped turn. The carbon atoms of peptide 2 are colored gold, whereas those of G(867) are colored purple. P, Pro; R, Arg; S, Ser; T, Thr; Y, Tyr.

Fig. 4

Proposed structure of the entire 3´UTR. (A) Diagram of the 3´UTR with color-coded U-C-hairpin-A-G CP-binding sites. (B) End-on view of the simulated 3´UTR. (C) View of the 3´UTR along its length. RNA is colored green; peptide is colored yellow.