A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons

Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z′-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency

The Highly Conserved Codon following the Slippery Sequence Supports −1 Frameshift Efficiency at the HIV-1 Frameshift Site

<div><p>HIV-1 utilises −1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating −1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the ‘intercodon’) contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules—eRF1 protein or a cognate suppressor tRNA—were able to access and decode the intercodon prior to −1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.</p></div

The identity of the intercodon influences −1 frameshift efficiency.

<p>Top: schematic representations of the base of the HIV-1 stem-loop, with the natural glycine codon and variants (left) and the substituted stop codon and variants showing the intercodon (grey) and any complementary stem-loop alterations (bold). The end of the slippery sequence is underlined. Below: frameshift efficiency of the natural intercodon and variants as assayed with the dual luciferase reporter system. GGG_AA and UGA_U indicate the intercodon (left part) and the modification to the complementary sequences in the stem-loop (right part). The mean ± standard error of the mean (SEM) for 18 replicates from three individual experiments in COS-7 cells is shown. ***<i>P</i> = < 0.001 compared to the GGG intercodon.</p

Specific intercodon suppressor tRNAs affect recoding efficiency.

<p>A. Readthrough at the test stop signals UAGAAG, UGAAAG and UAAAAG in HEK293T cells with cognate or non-cognate suppressor tRNAs (UAG [black], UGA [grey] or UAA [white]) and tRNA^Ser as a control (serine). The mean ± SEM for four replicates is shown. B. Effect of over-expressing cognate suppressor tRNA^UGA, non-cognate tRNA^UAG or control tRNA^Ser on human antizyme +1 frameshift efficiency. The mean ± SEM for four replicates is shown. C. Effect on −1 frameshift efficiency of the HIV-1 element when UAG (black) or UGA (grey) suppressor tRNAs or the control tRNA^Ser (white) are expressed. Frameshift efficiency was measured for the cognate, non-cognate, and control tRNAs. Note the change in scales between when the intercodon is the native GGG (left) and UGA or UGA with a complementary mutation restoring the stem (UGA_U, right). The mean ± SEM for four replicates (GGG) or six replicates (UGA and UGA_U) is shown. *<i>P</i> = < 0.05, **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001.</p

A modified model for −1 frameshifting in HIV-1.

<p>A. The first nucleotide (U) of the slippery sequence (blue), as part of the ‘BCX’ codon [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.ref026" target="_blank">26</a>], is positioned in the A site. The extended stem-loop is at the entrance to the ribosome, with the first nucleotide of the intercodon (orange) in the +13 position. B. The lower stem of the structure unwinds and allows the slippery sequence into the decoding centre. The stable upper stem-loop is positioned near the entry channel of the ribosome. Tension arising from resistance to unwinding may allow slippage from the A and P sites at this stage. C. After partial unwinding of the stable upper stem-loop, binding of tRNA^Gly to the intercodon, perhaps in a distorted state, competes with −1 frameshifting. D. The tRNA^Gly bound to the intercodon is decoded and translation proceeds in the 0 frame. This is the most common event, resulting in the translation of the Gag product. E. If the tRNA^Gly is not decoded, −1 frameshifting by the E and P site tRNAs occurs to relieve tension on the mRNA, resulting in the translation of Gag-Pol. An incoming tRNA^Arg is shown.</p

The effect of eRF1 depletion on termination and recoding.

<p>A. eRF1 transcript levels measured using quantitative real-time PCR. Results show eRF1 transcripts within RNA isolated from non-transfected HEK293T cells (‘none’), and eRF1 transcripts within RNA isolated from those transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The mean ± standard deviation (SD) for six replicates is shown. B. Immunoblot of eRF1 in protein extracts from non-transfected HEK293T cells (‘none’), or cells transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The ratios were calculated after normalisation to β-actin in each case and compared with the non-transfected control (1.0). Raw data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s001" target="_blank">S1 Fig.</a> C. Readthrough at a UGA test context (UGACAG). A dual luciferase construct with the two reporters in the same frame and the test stop signal separating them was co-transfected with the control and α-eRF1 shRNAs. Readthrough at the test stop signal was determined in each case (α-eRF1 and − control.) The mean ± SEM for 12 replicates from three individual experiments is shown. D. Effect of depletion of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for eight replicates is shown. E. Effect of eRF1 depletion on frameshifting with the native GGG intercodon and substituted stop codon (UGA or UAG). The mean ± SEM for a minimum of 10 replicates from at least two independent experiments is shown. A dotted line indicates the level of −1 PRF in the absence of any shRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.g002" target="_blank">Fig. 2</a>). **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001, n.s., not significant.</p

eRF1 over-expression influences frameshifting.

<p>A. eRF1 transcript levels measured using quantitative real-time PCR. Results show non-transfected HEK293T cells (‘none’), and those transfected with pcDNA-eRF1 vector or pcDNA3.1(+) with no insert (empty vector). The mean ± SD for six replicates is shown. B. Immunoblot of eRF1 expression in non-transfected HEK293T cells (‘none’), or cells transfected with pcDNA3.1(+) with no insert (empty vector) or pcDNA-eRF1 (eRF1). The ratios were calculated after normalisation to β-actin and comparison with the non-transfected control (1.0). Raw data are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s003" target="_blank">S3 Figs.</a> C. Effect of over-expression of eRF1 on readthrough at UGA test contexts. The mean ± SEM for four replicates (UGAAAG) or 12 replicates from three individual experiments (UGACUG) is shown. Cells were transfected with either empty vector (black) or pcDNA-eRF1 (grey). Values are shown above the bars for the strong stop signal UGAAAG. D. Effect of over-expression of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for six replicates is shown. E. Effect of over-expression of eRF1 on HIV-1 −1 frameshift efficiency with GGG intercodon and when it is substituted with a stop codon (UGA or UAG). The mean ± SEM for six replicates is shown. pcDNA (black), pcDNA-eRF1 (grey). *<i>P</i> = < 0.05, **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001, n.s., not significant.</p

Frameshift efficiency is dependent on the identity of the first nucleotide of the intercodon.

<p>Frameshift efficiency for the NGG (black) and NGA (grey) contexts show the mean ± SEM for 33 replicates from five individual experiments in HEK293T cells.</p

Mutation in the TCR alpha subunit constant gene (TRAC) leads to a human immunodeficiency disorder characterized by a lack of TCR alpha beta(+) T cells

Inherited immunodeficiency disorders can be caused by mutations in any one of a large number of genes involved in the function of immune cells. Here, we describe two families with an autosomal recessive inherited immunodeficiency disorder characterized by increased susceptibility to infection and autoimmunity. Genetic linkage studies mapped the disorder to chromosomal region 14q11.2, and a homozygous guanine-to-adenine substitution was identified at the last base of exon 3 immediately following the translational termination codon in the TCRα subunit constant gene (TRAC). RT-PCR analysis in the two affected individuals revealed impaired splicing of the mRNA, as exon 3 was lost from the TRAC transcript. The mutant TCRα chain protein was predicted to lack part of the connecting peptide domain and all of the transmembrane and cytoplasmic domains, which have a critical role in the regulation of the assembly and/or intracellular transport of TCR complexes. We found that T cells from affected individuals were profoundly impaired for surface expression of the TCRαβ complex. We believe this to be the first report of a disease-causing human TRAC mutation. Although the absence of TCRαβ(+) T cells in the affected individuals was associated with immune dysregulation and autoimmunity, they had a surprising level of protection against infection

Mass spectrometry identification of peptides containing the sites of frameshifting.

<p>The charge state (Charge), measured m/z value (m/z) and error of measurement in parts per million (Error; ppm) are given for the strongest detected peptide signal for each of the predicted frameshifting events. The scores for peptide identifications are given for both the SequestHT and Mascot search engines. The Mascot spectrum to peptide sequence assignments marked by an asterisk were not considered high confidence identifications, but also did not match any other sequence in the SwissProt sequence database (546,057 sequence entries) and therefore represent the most likely spectrum to sequence match. The peptide intensity is given as the area under the curve (peak area) for the extracted ion chromatogram of the strongest peptide signal of each frameshifting event and can be used as a rough estimation of peptide abundance. No CID: this low intensity signal was not selected for collision induced dissociation tandem mass spectrometry. The peptide detection is therefore based on high mass accuracy precursor mass measurement with an error of <1 ppm (+/−0.0016 Da).</p><p>Mass spectrometry identification of peptides containing the sites of frameshifting.</p