Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Abstract

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5' ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem-loop structures from one RNA form highly stringent cross-strand loop-loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions.

Fig. 1.

Conformational switch in the SL1-SL2 domain during retroviral RNA dimerization, defined by RNA SHAPE chemistry. (A) Structure of the MiDAS domain for the Moloney murine sarcoma virus in the monomeric starting state (9). RNA sequences that contribute to the SL1-SL2 conformational change are shown explicitly; other MiDAS structures are represented in gray. (B) SL1-SL2 domain conformation in the final dimer state. Regions in the RNA that undergo significant structural changes are emphasized in color. Dashed line (light blue) illustrates cross-strand base pairing in PAL2. Multiple systems are in use for naming structural features in gamma retroviral dimerization domains; we use the system introduced with the initial characterization of these elements (10).

Fig. 2.

SHAPE analysis of the MiDAS RNA in starting monomer-like (M) and final dimer (D) conformations and a simplified SL1-SL2 domain RNA in the final dimer state (D). (A) 2′-O-Adduct formation upon addition of NMIA (+) detected by primer extension. −, reactions omitting NMIA. Sequencing lanes (SEQ) were generated by dideoxy cytosine nucleotide incorporation; nucleotide positions are labeled with respect to NMIA lanes. (B) Quantitative histograms for NMIA reactivity. Columns are colored using the scheme shown in Fig. 1. Column heights report band intensities in the (+) NMIA reactions minus background.

Fig. 3.

Dimerization specificity of the SL1-SL2 domain in monomer-like versus final dimer states. (A and D) Monomer-like and final dimer states for the SL1-SL2 domain. Monomer contains a single inverted base pair to facilitate transcription (open letters). Control experiments show this base-pair change does not affect dimerization (data not shown). (B and E) Native gel analysis of RNA dimerization for wild type (CG/CG) and representative mutant sequences in monomer-like and final dimer conformations. M, monomer; D, dimer. (C and F) Binding curves for RNA dimerization in the monomer-like and final dimer states.

Fig. 4.

Architecture of the SL1–SL2 interaction in the final dimer conformation mapped by site-directed hydroxyl radical footprinting. (A) Fe(II)-BABE (open circle) mediated cleavage from nucleotide 310. (B) Fe(II)-ITE (filled circle) cleavage from nucleotide 336. Small spheres indicate RNA regions that were not monitored; the position of 5′ radiolabel on second RNA strand is indicated by an asterisk.

Fig. 5.

Refined model and two-step assembly of the SL1-SL2 domain. (A) Summary of 62 long-range intermolecular distance constraints used for structure refinement. Adjacent and repelling constraints are shown with solid and dashed lines, respectively. (B) Stereo image of the SL1-SL2 domain in the final dimer state. One monomer (red and magenta) is shown in a surface representation. The second monomer (blue and cyan) is illustrated as a backbone cartoon; bases are shown as cylinders. Cross-strand G-C pairs in the tetraloops are white. (C) Assembly of the high-affinity SL1-SL2 dimer via a stringent loop–loop intermediate.