Structure and Dynamics of the HIV-1 Frameshift Element RNA

Low, Justin; García‐Miranda, Pablo; Mouzakis, Kathryn D.; Gorelick, Robert J.; Butcher, Samuel E.; Weeks, Kevin M.

doi:10.1021/bi5004926

Cited by 32 publications

(39 citation statements)

References 40 publications

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“…3) but mainly differed in whether or not the slippery sequence was base paired. The weak thermodynamic stability of the slippery sequence helix is consistent with formamide titration data (14). Previously, we showed that the anchoring helix modulates frameshifting in vitro, with disruption of this helix causing an increase in frameshifting (6,14).…”

Section: Resultssupporting

confidence: 87%

“…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%

“…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). The role of the genomic secondary structure surrounding the HIV frameshift site has not been investigated in living cells.…”

mentioning

confidence: 97%

“…In vitro, the full secondary structure is required in order to stimulate frameshifting at the C1680U secondary slippery site (14). Using a dual-luciferase assay in rabbit reticulocyte lysate, we found that the C1680U mutation creates a functional slippery sequence that can be detected when the primary slippery site is knocked out by mutagenesis (14).…”

mentioning

confidence: 99%

“…Previous studies investigating the effect of the C1680U mutation on frameshifting utilized a truncated RNA construct that did not contain the full frameshift site secondary structure (16,17). In vitro, the full secondary structure is required in order to stimulate frameshifting at the C1680U secondary slippery site (14). Using a dual-luciferase assay in rabbit reticulocyte lysate, we found that the C1680U mutation creates a functional slippery sequence that can be detected when the primary slippery site is knocked out by mutagenesis (14).…”

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: 87%

supporting

confidence: 86%

mentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

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