An equilibrium-dependent retroviral mRNA switch regulates translational recoding

Abstract

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly1,2. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon3. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio4–8. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

“…An RNA pseudoknot structure (MoMLV-PK) is responsible for allowing translational readthrough of the MoMLV gag stop codon via termination suppression (reviewed in reference 4). Nuclear magnetic resonance-based structural studies demonstrate that, at physiological pH, the pseudoknot adopts a conformation permissive for stop codon readthrough in approximately 6% of MoMLV RNAs, whereas a nonpermissive conformation is observed in the remaining 94% (5). These ratios correlate well with the 20:1 Gag/Gag-Pol ratio reported for MLV (6).…”

Cryptic MHC class I-binding peptides are revealed by aminoglycoside-induced stop codon read-through into the 3′ UTR

“…It is possible that selectivity in read-through for conventional stop codons differs from that of PTCs. Moreover, numerous factors may influence translation fidelity, including nucleotides up to six bases away from the stop codon (33,34), translation factors (35), and local mRNA tertiary structure (36).…”

Retrotransposon Ty1 RNA contains a 5′-terminal long-range pseudoknot required for efficient reverse transcription

“…Indeed, for several viral pseudoknots, as long as their conformation and overall stability is maintained, the exact nucleotide sequence may not be important (Giedroc and Cornish 2009). On the other hand, it was recently demonstrated that the murine leukemia virus (MLV) pseudoknot regulates translational recoding by a protonation-dependent conformational switch that involves interactions of the stem with the loop (Houck-Loomis et al 2011). Although the Ty1 G6A mutant did not show substantial reactivity changes in the pseudoknot region, our clustering analysis demonstrated that this mutant is distinct from the WT in global structure.…”