HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Mouzakis, Kathryn D.; Lang, Andrew L.; Meulen, Kirk A. Vander; Easterday, Preston D.; Butcher, Samuel E.

doi:10.1093/nar/gks1254

Cited by 73 publications

(119 citation statements)

References 83 publications

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“…2E and F). These results are highly similar to previously described in vitro data (6). As expected, no frameshifting was detected when the slippery site was mutated to a nonslippery sequence ( Fig.…”

Section: Resultssupporting

confidence: 92%

“…Consistent with this idea, Brakier-Gingras and coworkers found that decreased translation initiation of HIV-1 mRNA resulted in increased frameshifting (13). Additionally, we found that disruption of the genomic secondary structure by mutagenesis led to increased frameshifting in vitro (6,14). SHAPE (selective 2-hydroxyl acylation analyzed by primer extension) data indicate that the genomic secondary structure is remodeled during translation, as regions of the RNA are unfolded and sequestered by the ribosome (14).…”

supporting

confidence: 86%

“…2A and B; M5). Frameshift efficiency was plotted as a function of overall stability and local (3-bp) stability using previously described free energy values (6). The data from the mutants showed a modest correlation as a function of overall RNA stability (R 2 ϭ 0.67); however, we observed a strong correlation (R 2 ϭ 0.95) between frameshifting and the thermodynamic stability of the first 3 bp (Fig.…”

Section: Resultsmentioning

confidence: 78%

“…As the mRNA entrance tunnel is only wide enough to accommodate single-stranded mRNA, the first few base pairs of the stem-loop are expected to be engaged with ribosomal surface helicase-like proteins S3 and S9 (9,10) at the time of frameshifting. Consistent with this, our previous experiments used a dual-luciferase assay in rabbit reticulocyte lysate to show that the local thermodynamic stability of the first 3 bp in the frameshift site stem-loop is highly correlated to frameshift efficiency in vitro (6). However, it is not known if a similar thermodynamic correlation exists within cells during viral replication.…”

mentioning

confidence: 77%

“…The slippery site allows near-cognate repairing of the A and P site tRNA anticodons in the Ϫ1 reading frame. The 8-nucleotide (nt) spacing between the slippery site and the stem-loop corresponds to the length of mRNA required to span the distance from the ribosomal A site to the mRNA entrance tunnel on the surface of the ribosome (6,8). As the mRNA entrance tunnel is only wide enough to accommodate single-stranded mRNA, the first few base pairs of the stem-loop are expected to be engaged with ribosomal surface helicase-like proteins S3 and S9 (9,10) at the time of frameshifting.…”

mentioning

confidence: 99%

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Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

García‐Miranda

Becker

Benner

et al. 2016

J Virol

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Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus-or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gagpolymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCEHIV, like many retroviruses, utilizes a ؊1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies. The genome of human immunodeficiency virus type 1 (HIV-1), like that of other retroviruses, has three genes that encode the structural proteins Gag, polymerase (Pol), and Env. The expression of the gag gene results in the synthesis of the Gag precursor protein, p55, which is subsequently processed by the viral protease to release the mature Gag proteins p17 (matrix protein), p24 (capsid), p15 (nucleocapsid), and p6 (late domain) and two so-called spacer peptides (SP) that flank p15, namely, p2 (SP1) and p1 (SP2), respectively (1). The synthesis of Gag precursor protein alone is sufficient for the assembly and release of noninfectious virus-like particles (VLPs) (2). The pol gene codes for the p160 polyprotein, which is su...

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Section: Resultssupporting

confidence: 92%

supporting

confidence: 86%

Section: Resultsmentioning

confidence: 78%

mentioning

confidence: 77%

mentioning

confidence: 99%

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Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

García‐Miranda

Becker

Benner

et al. 2016

J Virol

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A U⋅U Pair‐to‐U⋅C Pair Mutation‐Induced RNA Native Structure Destabilisation and Stretching‐Force‐Induced RNA Misfolding

et al. 2015

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Little is known about how a non‐Watson–Crick pair affects the RNA folding dynamics. We studied the effects of a U⋅U‐to‐U⋅C pair mutation on the folding of a hairpin in human telomerase RNA. The ensemble thermal melting of the hairpins shows an on‐pathway intermediate with the disruption of the internal loop structure containing the U⋅U/U⋅C pairs. By using optical tweezers, we applied a stretching force on the terminal ends of the hairpins to probe directly the non‐nearest‐neighbour effects upon the mutations. The single U⋅U to U⋅C mutations are observed to 1) lower the mechanical unfolding force by approximately 1 picoNewton (pN) per mutation without affecting the unfolding reaction transition‐state position (thus suggesting that removing a single hydrogen bond affects the structural dynamics at least two base pairs away), 2) result in more frequent misfolding into a small hairpin at approximately 10 pN and 3) shift the folding reaction transition‐state position towards the native hairpin structure and slightly increase the mechanical folding kinetics (thus suggesting that untrapping from the misfolded state is not the rate‐limiting step).

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Dynamic Motions of the HIV-1 Frameshift Site RNA

Mouzakis

Dethoff

Tonelli

et al. 2015

Biophysical Journal

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The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (β = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions.

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HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Cited by 73 publications

References 83 publications

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

A U⋅U Pair‐to‐U⋅C Pair Mutation‐Induced RNA Native Structure Destabilisation and Stretching‐Force‐Induced RNA Misfolding

Dynamic Motions of the HIV-1 Frameshift Site RNA

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