30 research outputs found

    Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation

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    <div><p>The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from <b>D</b>efective <b>Ri</b>bosomal <b>P</b>roducts (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.</p> </div

    Positional biases of predicted binders for 12 HLA supertypes.

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    <p>For each supertype, 9-mer peptide binding predictions were carried out and ratios of probability masses of predicted ‘binders’ and ‘non-binders’ were calculated. Peptide binding predictions were made for alleles belonging to each supertype, using SMM<sup>PMBEC</sup> method. All possible 9-mer peptides were generated from a set of viral proteins that contain at least one tested peptide from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-t001" target="_blank">Table 1</a>. Relationships between HLA molecules and supertypes are provided in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi.1002884-Sidney1" target="_blank">[11]</a>.</p

    MHC class I restricted T-cell epitopes retrieved from the Immune Epitope Database for viral species.

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    <p>For each organism, total number of tested peptides as well as numbers of those with positive and negative assay outcomes are shown. A total of 93 viruses were studied. For brevity, 20 viruses with the highest number of tested peptides are shown in the table.</p

    Estimating positional bias of epitopes from conservation data.

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    <p>(A) Bootstrap sampling of conservation scores for positive and negative peptides are shown as two boxplots placed next to each other. The bins used are of variable lengths to ensure a sufficient count in each bin. Each bin contains ∼20% of data points. Middle positions of bins are indicated on the x-axis. The difference between the means of the two conservation score distributions is statistically significant (Welch's t-test; one-sided; p-value = 5.9×10<sup>−6</sup>). (B) Estimating probability ratios from the conservation score distributions. This is simply taking a ratio of positive and negative peptide probabilities as a function of a conservation score. Confidence intervals are derived from bootstrap sampling. (C) Estimated probability ratios as a function of normalized position, using the mapping shown in the second panel. As input, distributions of means of conservation scores shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-g003" target="_blank">Fig. 3</a> were used. (D) Positional bias curves derived from observed normalized positions (black) and conservation scores (gray).</p

    Distributions of conservation scores of peptides with positive and negative T-cell assay results for viruses in the context of immunization with peptides.

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    <p>The difference between the two distributions is not statistically significant (Welch's t-test; one-sided; p-value = 0.62).</p

    Positional bias of conservation for viral proteins.

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    <p>For each viral protein which also contained at least one tested peptide from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-t001" target="_blank">Table 1</a>, conservation scores at the residue-level were calculated using Rate4Site <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi.1002884-Mayrose1" target="_blank">[26]</a>. Residue positions were converted into normalized positions and corresponding conservation scores were binned (5 bins of equal size). Higher conservation score indicates higher degree of conservation. Conservation scores were normalized for each protein (i.e. mean = 0; sd = 1). One thousand bootstrapping over proteins was used to estimate confidence intervals for the means of conservation scores. Each box covers 25<sup>th</sup> and 75<sup>th</sup> percentiles. Whiskers extend out from each box 1.0 times the interquartile range.</p

    Leu-tRNA, Lys-tRNA and Ser-tRNA deacylation is coupled to translation.

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    <p>A. Kinetic of [<sup>3</sup>H]-Leu deacylation on tRNAs extracted from HeLa cells treated or not with sodium arsenite. B. Same as “A” with [<sup>3</sup>H]-Lys deacylation. C. Same as “A” with [<sup>3</sup>H]-Ser deacylation.</p

    Cys-tRNA deacylation is uncoupled from translation.

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    <p>A. Kinetics of [<sup>35</sup>S]-Met charging on tRNAs with or without pre-treating HeLa cells with sodium arsenite. The same samples were both analyzed by beta counter and loaded on a native acid gel. B. Same as “A” with [<sup>35</sup>S]-Cys charging. C. Kinetics of [<sup>35</sup>S]-Met deacylation on tRNAs extracted from HeLa cells pretreated or not with sodium arsenite. This panel shows two different graph representations: “raw” data using Cpm/µg of total RNA and representing one experiment or calibrated data defining time 0 as 100% of [<sup>35</sup>S]-Met charged tRNAs and averaging 2 distinct experiments. The same samples were both analyzed by beta counter and loaded on native acid gels. D. Same as “A” with [<sup>35</sup>S]-Cys deacylation.</p

    MRS and CRS define two distinct cytosolic compartments.

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    <p>A. Immunofluorescence staining of HeLa cells. MRS (green) and CRS (red) only partially co-localize. Bar scale: 10 µm. B. Immunoblot of sequential HeLa cells extractions. Cells were first extracted with digitonin (DSC: digitonin soluble compartment), then washed and extracted with NP-40 (DRC: digitonin resistant compartment). Membranes were blotted for MRS, CRS and ribosomal P proteins. C. HeLa cells were pre-treated for 2 h with MG132 or for 30 min with either arsenite or pactamycin. Then cells were labeled with [<sup>35</sup>S]-Cys or [<sup>35</sup>S]-Met. Half of each sample was used to extract total proteins and cpm were counted. 100% cpm was determined using untreated HeLa cells. For [<sup>35</sup>S]-Met: 100% = 982785 cpm. For [<sup>35</sup>S]-Cys: 100% = 371828 cpm. D. The other half of each sample from “C” was used. Total RNAs were extracted and cpm counted. 100% cpm was determined using untreated HeLa cells. For [<sup>35</sup>S]-Met: 100% = 288248 cpm. For [<sup>35</sup>S]-Cys: 100% = 69183 cpm. E. The same samples used in “D” were loaded on acid gels to visualize either [<sup>35</sup>S]-Met-tRNAs or [<sup>35</sup>S]-Cys-tRNAs. The two bands for [<sup>35</sup>S]-Met-tRNAs correspond to the mitochondrial tRNA<sup>Met</sup> and the nuclear encoded tRNA<sup>Met</sup>.</p

    Kinetics of anti-VV IL-10<sup>+</sup> CD8<sup>+</sup> T cell responses.

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    <p><b>A)</b> Viral titers after epicutanous infection of the ear as determined by plaque assay at indicated day p.i. N = 3 <b>B)</b> Time course of total CD4<sup>+</sup> (green lines) and CD8<sup>+</sup> T cells (blue lines) in the ear skin. N = 4. <b>C)</b> IL-10<sup>+</sup>CD4<sup>+</sup> (green) and IL-10<sup>+</sup>CD8<sup>+</sup> (blue) T cells. N = 4. <b>D)</b> Percentage of IL-10gfp<sup>+</sup> CD8<sup>+</sup> T cells of total CD8<sup>+</sup> T cells in the ear skin (circle), draining lymph node (dLN, square), or spleen (triangle). <b>E)</b> Percentage of IL-10gfp<sup>+</sup> CD4<sup>+</sup> T cells of the total CD4 T cells as in (D). Error bars = SEM. **** = P <0.0001.</p
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