Stacked area charts of M_163-177 and NS2_31-45 synthetic peptides digested with purified ERAPs.

<p>M_163-177 (panels A-D) and NS2_31-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.</p

MALDI-MS analysis of the M_163-177 synthetic peptide digested with purified ERAPs.

<p>M_163-177 substrate (Panel A) was digested overnight with ERAP as follow: ERAP1 at an E/S ratio of 1:800 (panel B), ERAP2 at an E/S ratio of 1:800 (panel C), both ERAP1 and 2 at an E/S ratio of 1:800 for each enzyme (panel D), and both ERAP1 and 2 at an E/S ratio of 1:1600 for each enzyme (panel E). MALDI-TOF analysis of digestions detected M_163-177 substrate and N-trimmed peptides, as well as several adducts and neutral loss of peptides (marked by an arrow or asterisk, respectively). The <i>m</i>/<i>z</i> range represented in the <i>x</i>-axis is 1000-1850. The <i>m</i>/<i>z</i> position and length of each possible N-trimmed peptide is indicated with an arrow at the base of the figure.</p

Concerted <i>In Vitro</i> Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

<div><p>In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These <i>in vitro</i> data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.</p> </div

HLA stabilization assay with monosubstituted Ala analogs of HRSV NP _184-194 and NP _195-205 synthetic ligands.

a<p>Data are expressed as fluorescence index when peptides were used at 200 µM ± S.D. The results show the mean of three to five independent experiments. All data show significant P values (P<0.01) versus the negative control CMV pp65, except the two values underlined. In addition, the fluorescence index of A1-NP _184-194 with B*2703 subtype or A11-NP _195-205 peptide with B*2702 subtype (marked in bold) also shows significant P values (P<0.01) versus either the negative control or the NP _184-194 and NP _195-205 peptides, respectively.</p><p>HLA stabilization assay with monosubstituted Ala analogs of HRSV NP _184-194 and NP _195-205 synthetic ligands.</p

Stacked area charts of the N_95-109 synthetic peptide digested with purified ERAPs.

<p>The N_95-109 synthetic peptide (sequence indicated at the top of the figure and the 9-mer identified by MS is bolded), was digested at different times with ERAP as follows: ERAP1 at an E/S ratio of 1:200 (panel A), ERAP2 at an E/S ratio of 1:200 (panel B), both ERAP1 and 2 at an E/S ratio of 1:200 (panel C), and both ERAP1 and 2 at an E/S ratio of 1:400 (panel D). The results depicted as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079596#pone-0079596-g002" target="_blank">Figure 2</a> are the mean values of two independent experiments.</p

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein

<div><p>Background</p><p>The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne <i>Alphavirus</i> of the <i>Togaviridae</i> family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized.</p><p>Methodology/Principal findings</p><p>By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells.</p><p>Conclusions/Significance</p><p>Our data have implications not only for the identification of novel <i>Alphavirus</i> and <i>Togaviridae</i> antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.</p></div

Stacked area charts of M_163-177 and NS2_31-45 synthetic peptides digested with purified ERAPs.

<p>M_163-177 (panels A-D) and NS2_31-45 (panels E-H) (sequences are indicated at the top of the figure and the respective HLA ligands identified by MS are bolded) synthetic peptides were digested at different times with ERAP enzymes as follows: ERAP1 at an E/S ratio of 1:800 (panels A and E), ERAP2 at an E/S ratio of 1:800 (panels B and F), both ERAP1 and 2 at an E/S ratio of 1:800 (panels C and G), and both ERAP1 and 2 at an E/S ratio of 1:1600 (panels D and H). The intensity peaks obtained by MALDI-TOF analysis for all peptides at each time point were added and taken as 100% for each time point and depicted. The different N-end trimming products detected are named in their respective region. The results depicted are the mean values of three or four independent experiments.</p

CHIKV 6K peptide specificity of HLA-A*0201-restricted CD8⁺ T cell lines.

<p>Mouse HLA-A*0201⁺ DC pre-pulsed with 10⁻⁶ M of indicated CHIKV 6K synthetic peptide were analyzed by ICS for CD8⁺ T cell activation with CHIKV peptide-specific CD8⁺ T cells from HLA-A*0201-transgenic mice immunized with rVACV-CHIKV and restimulated <i>in vitro</i> with the appropriate CHIKV 6K synthetic peptide. Graph data shown as mean ± SD of four independent experiments (*** P <0.001). Representative ICS panels with non-specific or CHIKV peptide-specific CD8⁺ T cell lines are depicted beneath the graphs. The percentages of IFNγ-expressing CD8⁺ T cells are indicated in each dot plot.</p

Diversity of proteases and processing pathway involved in CHIKV 6K_51-59 epitope presentation.

<p>The model shows the components of the antigen presentation pathway proposed for the CHIKV 6K_51-59 epitope. Stop-transfer signals are indicated by rectangular blocks, and signal sequences by dashed cylinders. Subcellular organelles are shown as colored boxes: cytosol (yellow), ER (blue), trans-Golgi network (mauve) and lysosomes (green). CHIKV proteins are capsid (CP, maroon), p62 (yellow), 6K (green), E1 (peach), E2 (blue), and E3 (brown). The CHIKV 6K_51-59 epitope is depicted in red. The role of the distinct proteases is deduced from CD8⁺ T cell sensitivity to the various inhibitors, except for the signal peptidase, whose role was described in the generation of the Alphavirus 6K protein [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.ref059" target="_blank">59</a>].</p

Summary of the relative affinity of HRSV ligands for different HLA-B27 subtypes.

a<p>The new HLA-B27 anchor motifs for efficient HLA binding are underlined.</p>b<p>+, ++, +++ and ++++ indicate EC₅₀ values>200 µM, 200-61 µM, 60-20 µM, and <20 µM, respectively. – indicates no statistical difference in fluorescence index compared to the negative control. All positive EC₅₀ data show significant P values (P<0.01) versus the negative control.</p><p>Summary of the relative affinity of HRSV ligands for different HLA-B27 subtypes.</p