Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

Abstract

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

Fig. 1.

Structure of the MuLV dimerization domain in the dimer state. (A) Secondary structure. The two RNA strands are shown in black and gray. Major structural elements are labeled. Protein-binding sites at UCUG tandem sequences identified in this work are boxed. (B) Local structure at each recognition site based on binding experiments (Fig. 5) and on prior work showing extensive helix packing and tertiary structure in this region of the MuLV RNA (38, 45).

Fig. 5.

Defining the minimal RNA binding motif for Gag. (A and B) Representative analysis of Gag binding to the PAL1-RS and PAL2-RS RNAs. K_d’s for PAL1-RS and PAL2-RS (constructs 1-1 and 2-1) are 120 and 110 nm, respectively. (C and D) Effect of point mutations on Gag binding to PAL1-RS and PAL2-RS. Mutated sites relative to the native sequence are shown in red. Binding affinities for the mutants are reported as K_rel, equal to ; larger values indicate weaker binding. K_rel values larger than 2 are emphasized in red.

Fig. 2.

Structural differences in the Ψ domain as a function of genomic RNA state. (A) SHAPE reactivity histograms for the intact in virio (red), AT-2 treated (dark blue), and ex virio (light blue) vRNA. Broken lines indicate a small number of nucleotides that were not analyzed due to high background. (B) Difference plot calculated by subtracting the ex virio (light blue columns) or AT-2 treated (dark blue columns) experiments from the in virio data. Positive and negative amplitudes indicate nucleotides that show greater or lesser flexibility, respectively, in virio as compared to the other two states. The two strongest sites of increased reactivity in the AT-2 treated and ex virio states are emphasized with bold lines.

Fig. 3.

Identification of specific protein binding sites in the MuLV Ψ region. Protection factors correspond to (I_- - I₊)/I₊, where I₊ and I_- are SHAPE reactivities in the presence and absence of protein, respectively. Conserved reactivity patterns at UCUG sequences are outlined in red. Nucleotides that were unreactive both before and after protein addition (reactivity less than 0.15 SHAPE units) are omitted.

Fig. 4.

Gag binding to the full-length MuLV dimerization domain. Data points, shown as the mean and standard deviation for four replicates, were fit to an equation for two independent binding events; R² is ≥0.97 in all cases. K_app,1 for the native, M1, M2, and M1M2 RNAs are 6 ± 2, 11 ± 3, 12 ± 3, and 35 ± 12 nM, respectively.

Fig. 6.

Normalized encapsidation efficiencies for MuLV-derived pBabe-Luc RNAs containing native sequence and mutant Ψ domains. Geometric means and standard deviations are shown.