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• W2151878384 abstract "Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response. Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response. The recognition of short viral peptides associated with human histocompatibility complex (human leukocyte antigen (HLA) 1The abbreviations used are:HLAhuman leukocyte antigenAbantibodyB27-C1RHMy2.C1R transfected with HLA-B*2705CTLcytotoxic T lymphocyteEC50half-maximal effective concentrationHRSVhuman respiratory syncytial virusMHCmajor histocompatibility complexTAPtransporter associated to antigen processingγLCmicrocapillary LCSHsmall hydrophobic.) class I molecules on the cell surface allows cytotoxic T lymphocytes (CTLs) to recognize and kill virus-infected cells (1.Shastri N. Schwab S. Serwold T. Producing nature's gene-chips: the generation of peptides for display by MHC class I molecules.Annu. Rev. Immunol. 2002; 20: 463-493Crossref PubMed Scopus (246) Google Scholar). These peptides are generated by proteolytic processing of newly synthesized viral proteins in the cytosol by the combined action of proteasomes, ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), and in some cases other peptidases (2.Del-Val M. López D. Multiple proteases process viral antigens for presentation by MHC class I molecules to CD8+ T lymphocytes.Mol. Immunol. 2002; 39: 235-247Crossref PubMed Scopus (46) Google Scholar). This degradation of viral proteins generates peptides of 8–11 residues that are translocated to the endoplasmic reticulum lumen by transporters associated with antigen processing. These short peptides then assemble with the HLA class I heavy chain and β2-microglobulin. Usually, two major anchor residues in the antigenic peptide, at position 2 and the C terminus (3.Parker K.C. Bednarek M.A. Coligan J.E. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.J. Immunol. 1994; 152: 163-175PubMed Google Scholar, 4.Rammensee H. Bachmann J. Emmerich N.P. Bachor O.A. Stevanoviæ S. SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics. 1999; 50: 213-219Crossref PubMed Scopus (1914) Google Scholar), must be deeply accommodated into specific pockets of the antigen recognition site of the HLA class I molecule to stabilize the nascent complexes (5.Bjorkman P.J. Saper M.A. Samraoui B. Bennett W.S. Strominger J.L. Wiley D.C. Structure of the human class I histocompatibility antigen, HLA-A2.Nature. 1987; 329: 506-512Crossref PubMed Scopus (2721) Google Scholar, 6.Bjorkman P.J. Saper M.A. Samraoui B. Bennett W.S. Strominger J.L. Wiley D.C. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.Nature. 1987; 329: 512-518Crossref PubMed Scopus (1812) Google Scholar) and allow for their subsequent transport to the cell membrane where they are exposed for CTL recognition (7.York I.A. Goldberg A.L. Mo X.Y. Rock K.L. Proteolysis and class I major histocompatibility complex antigen presentation.Immunol. Rev. 1999; 172: 49-66Crossref PubMed Scopus (186) Google Scholar). human leukocyte antigen antibody HMy2.C1R transfected with HLA-B*2705 cytotoxic T lymphocyte half-maximal effective concentration human respiratory syncytial virus major histocompatibility complex transporter associated to antigen processing microcapillary LC small hydrophobic. Human respiratory syncytial virus (HRSV) (8.Collins P.L. Chanock R.M. Murphy B.R. Respiratory syncytial virus.in: Knipe D.M. Howley P.M. Fields Virology. 5th Ed. Lippincott Williams and Wilkins, Philadelphia2007: 1443-1486Google Scholar), a member of the Paramyxoviridae family, is the single most important cause of bronchiolitis and pneumonia in infants and young children (9.Hall C.B. Respiratory syncytial virus and parainfluenza virus.N. Engl. J. Med. 2001; 344: 1917-1928Crossref PubMed Scopus (908) Google Scholar, 10.Shay D.K. Holman R.C. Roosevelt G.E. Clarke M.J. Anderson L.J. Bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among US children, 1979–1997.J. Infect. Dis. 2001; 183: 16-22Crossref PubMed Scopus (393) Google Scholar, 11.Thompson W.W. Shay D.K. Weintraub E. Brammer L. Cox N. Anderson L.J. Fukuda K. Mortality associated with influenza and respiratory syncytial virus in the United States.JAMA. 2003; 289: 179-186Crossref PubMed Scopus (2972) Google Scholar). Infections of this virus occur in people of all ages, but although usually mild infections are reported in healthy adults, HRSV poses a serious health risk in immunocompromised individuals (12.Wendt C.H. Hertz M.I. Respiratory syncytial virus and parainfluenza virus infections in the immunocompromised host.Semin. Respir. Infect. 1995; 10: 224-231PubMed Google Scholar, 13.Ison M.G. Hayden F.G. Viral infections in immunocompromised patients: what's new with respiratory viruses?.Curr. Opin. Infect. Dis. 2002; 15: 355-367Crossref PubMed Scopus (142) Google Scholar) and in the elderly (14.Han L.L. Alexander J.P. Anderson L.J. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden.J. Infect. Dis. 1999; 179: 25-30Crossref PubMed Scopus (247) Google Scholar, 15.Falsey A.R. Hennessey P.A. Formica M.A. Cox C. Walsh E.E. Respiratory syncytial virus infection in elderly and high-risk adults.N. Engl. J. Med. 2005; 352: 1749-1759Crossref PubMed Scopus (1310) Google Scholar). The single-stranded, negative-sense RNA genome of this enveloped virus codes for 11 proteins. Although the immune mechanism involved in HRSV disease and protection is not well understood, specific CD8+ T lymphocytes are required for the clearance of virus-infected cells (16.Anderson L.J. Heilman C.A. Protective and disease-enhancing immune responses to respiratory syncytial virus.J. Infect. Dis. 1995; 171: 1-7Crossref PubMed Scopus (80) Google Scholar). Previously, several HRSV epitopes restricted by different HLA class I molecules were identified using CTLs from seropositive individuals (17.Brandenburg A.H. de Waal L. Timmerman H.H. Hoogerhout P. de Swart R.L. Osterhaus A.D. HLA class I-restricted cytotoxic T-cell epitopes of the respiratory syncytial virus fusion protein.J. Virol. 2000; 74: 10240-10244Crossref PubMed Scopus (41) Google Scholar, 18.Rock M.T. Crowe Jr., J.E. Identification of a novel human leucocyte antigen-A*01-restricted cytotoxic T-lymphocyte epitope in the respiratory syncytial virus fusion protein.Immunology. 2003; 108: 474-480Crossref PubMed Scopus (45) Google Scholar, 19.Venter M. Rock M. Puren A.J. Tiemessen C.T. Crowe Jr., J.E. Respiratory syncytial virus nucleoprotein-specific cytotoxic T-cell epitopes in a South African population of diverse HLA types are conserved in circulating field strains.J. Virol. 2003; 77: 7319-7329Crossref PubMed Scopus (34) Google Scholar, 20.Heidema J. de Bree G.J. De Graaff P.M. van Maren W.W. Hoogerhout P. Out T.A. Kimpen J.L. van Bleek G.M. Human CD8(+) T cell responses against five newly identified respiratory syncytial virus-derived epitopes.J. Gen. Virol. 2004; 85: 2365-2374Crossref PubMed Scopus (58) Google Scholar, 21.Terrosi C. Di Genova G. Savellini G.G. Correale P. Blardi P. Cusi M.G. Immunological characterization of respiratory syncytial virus N protein epitopes recognized by human cytotoxic T lymphocytes.Viral Immunol. 2007; 20: 399-406Crossref PubMed Scopus (12) Google Scholar). However, these experiments were performed with synthetic peptides against individual proteins. In contrast, only one published study attempted to elucidate the nature and diversity of the possible array of HRSV ligands restricted by individual HLA molecules (22.Meiring H.D. Soethout E.C. Poelen M.C. Mooibroek D. Hoogerbrugge R. Timmermans H. Boog C.J. Heck A.J. de Jong A.P. van Els C.A. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.Mol. Cell. Proteomics. 2006; 5: 902-913Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In this study, virus-infected cells were cultured with stable, isotope-labeled amino acids, which were expected to act as anchor residues for the HLA allele of interest. The MHC molecules were then immunoprecipitated, and mass spectrometry analysis was performed. This study identified one HRSV ligand for each of the HLA-A2 and -B7 class I molecules (22.Meiring H.D. Soethout E.C. Poelen M.C. Mooibroek D. Hoogerbrugge R. Timmermans H. Boog C.J. Heck A.J. de Jong A.P. van Els C.A. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.Mol. Cell. Proteomics. 2006; 5: 902-913Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, is only one HRSV ligand restricted by a single HLA molecule exposed on the cell membrane surface as suggested by this study? Conversely, could a particular HLA molecule bind several ligands of this small virus simultaneously? To answer these questions, we compared HLA-B27 ligands isolated from large amounts of healthy or HRSV-infected cells without any methodological bias (selection of individual protein, use of HLA consensus scoring algorithms, etc.). This analysis demonstrated the existence of diverse, naturally processed HLA-B27 ligands from six different HRSV proteins in infected cells. B27-C1R is a transfectant (23.Calvo V. Rojo S. López D. Galocha B. López de Castro J.A. Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar) of the human lymphoid cell line HMy2.C1R (C1R) that expresses its endogenous HLA class I antigens at a low level (24.Storkus W.J. Howell D.N. Salter R.D. Dawson J.R. Cresswell P. NK susceptibility varies inversely with target cell class I HLA antigen expression.J. Immunol. 1987; 138: 1657-1659PubMed Google Scholar, 25.Zemmour J. Little A.M. Schendel D.J. Parham P. The HLA-A,B “negative” mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon.J. Immunol. 1992; 148: 1941-1948PubMed Google Scholar). RMA-S is a transporter associated to antigen processing (TAP)-deficient murine cell line that expresses the mouse H-2b haplotype (26.Ljunggren H.G. Kärre K. Host resistance directed selectively against H-2-deficient lymphoma variants. Analysis of the mechanism.J. Exp. Med. 1985; 162: 1745-1759Crossref PubMed Scopus (642) Google Scholar). The RMA-S transfectant cells expressing HLA-B27 were described previously (27.Villadangos J.A. Galocha B. López de Castro J.A. Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity.J. Immunol. 1994; 152: 2317-2323PubMed Google Scholar). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5 γm β-mercaptoethanol. The Abs used in this study were the polyclonal FITC-anti-HRSV Ab (which recognizes HRSV F and G proteins) (Chemicon International, Single Oak Drive Temecula, CA), the monoclonal W6/32 Ab (which is specific for a monomorphic HLA-A, -B, -C determinant) (28.Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1583) Google Scholar), and the ME1 Ab (which is specific for HLA-B27, -B7, -Bw22) (29.Ellis S.A. Taylor C. McMichael A. Recognition of HLA-B27 and related antigen by a monoclonal antibody.Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (197) Google Scholar). Peptides were synthesized in a peptide synthesizer (model 433A, Applied Biosystems, Foster City, CA) and purified by reverse-phase HPLC. The correct molecular mass of peptides was established with MALDI-TOF MS, and the correct composition of HRSV peptides was determined by quadrupole ion trap micro-HPLC. HLA-bound peptides were isolated from 4 × 1010 healthy or HRSV-infected B27-C1R transfectant cells. Cells were lysed in 1% Igepal CA-630 (Sigma), 20 mm Tris/HCl buffer, 150 mm NaCl, pH 7.5 in the presence of a protease inhibitor mixture. HLA-peptide complexes were isolated by affinity chromatography of the soluble fraction with the W6/32 monoclonal Ab. HLA-bound peptides were eluted at room temperature with 0.1% aqueous TFA and concentrated with a Centricon 3 column (Amicon, Beverly, MA) as described previously (30.Cragnolini J.J. López de Castro J.A. Identification of endogenously presented peptides from Chlamydia trachomatis with high homology to human proteins and to a natural self-ligand of HLA-B27.Mol. Cell. Proteomics. 2008; 7: 170-180Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31.Cragnolini J.J. Garcia-Medel N. López de Castro J.A. Endogenous processing and presentation of T-cell Epitopes from chlamydia trachomatis with relevance in HLA-B27-associated reactive arthritis.Mol. Cell. Proteomics. 2009; 8: 1850-1859Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Peptide mixtures recovered after the ultrafiltration step were concentrated with Micro-Tip reverse-phase columns (C18, 200 γl, Harvard Apparatus, Holliston, MA). Each C18 tip was equilibrated with 80% acetonitrile in 0.1% TFA, washed with 0.1% TFA, and then loaded with the peptide mixture. The tip was then washed with an additional volume of 0.1% TFA, and the peptides were eluted with 80% acetonitrile in 0.1% TFA. Peptide samples were then concentrated to about 18 γl using vacuum centrifugation. Recovered HLA class I peptides were analyzed in three different HPLC runs by γLC-MS/MS using an Orbitrap XL mass spectrometer (Thermo Electron, San Jose, CA) fitted with a capillary HPLC column (Eksigent, Dublin, CA). The peptides were resolved on homemade Reprosil C18 capillary columns (75-γm inner diameter) (32.Ishihama Y. Rappsilber J. Andersen J.S. Mann M. Microcolumns with self-assembled particle frits for proteomics.J. Chromatogr. A. 2002; 979: 233-239Crossref PubMed Scopus (253) Google Scholar) with a 7–40% acetonitrile gradient for 2 h in the presence of 0.1% formic acid. The seven most intense masses that exhibited single, double, and triple charge states were selected for fragmentation from each full mass spectrum by CID. Pep-Miner (33.Beer I. Barnea E. Ziv T. Admon A. Improving large-scale proteomics by clustering of mass spectrometry data.Proteomics. 2004; 4: 950-960Crossref PubMed Scopus (167) Google Scholar) was used for peak list generation of the γLC-MS/MS data. The peaks were identified using multiple search engines: Pep-Miner, Proteome Discoverer 1.0 SP1 (Thermo) combining the results of Sequest 3.31 and Bioworks Browser 3.3.1 SP1 (ThermoFisher) (34.Eng J. McCormack A. Yates J. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 2009; 5: 976-989Crossref Scopus (5315) Google Scholar), and Mascot (server 2.2, Matrix Science) (35.Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6661) Google Scholar) using the human and the virus parts of the NCBI database (June 2008) including 574,003 proteins. The search was not limited by enzymatic specificity, the peptide tolerance was set to 0.01 Da, and the fragment ion tolerance was set to 0.5 Da. This search was not limited by any restriction bias (selection of individual protein, use of HLA consensus scoring algorithms, etc.). Identified peptides were selected if the following criteria exist: Pep-Miner score above 75, Mascot score above 20, Sequest Xcorr above 2, P(pep) less than 1 × 10−4 with Bioworks Browser, Proteome Discoverer score higher than 20, and mass accuracy of 0.005 Da (see Table I). When the MS/MS spectra fitted more than one peptide, only the highest scoring peptide was analyzed.Table ISummary of HRSV ligands detected by MS/MS analysisNominal massaMass of monoisotopic ion in amu.Experimental massaMass of monoisotopic ion in amu.ΔMassbDifference between nominal and experimentally detected monoisotopic ions.m/zSequencecThe HLA-B27 anchor motifs are underlined.ProteinPosition614.299614.2970.0022+HRQDINGKEMNucleoprotein100–109409.868409.8680.0003+HRQDINGKEMNucleoprotein100–109672.862672.8580.0042+RRANNVLKNEMNucleoprotein184–194444.278444.2770.0013+KRYKGLLPKDINucleoprotein195–205488.766488.7640.0022+SRSALLAQMMatrix76–84536.809536.8070.0022+VRNKDLNTLMatrix169–177525.770525.7690.0012+GRNEVFSNKPolymerase2089–2097389.924389.9220.0023+KRLPADVLKKMatrix 2-22k150–159445.563445.5620.0013+LRNEESEKMAKPhosphoprotein198–208404.893404.8910.0023+HRFIYLINHNon-structural protein 237–45a Mass of monoisotopic ion in amu.b Difference between nominal and experimentally detected monoisotopic ions.c The HLA-B27 anchor motifs are underlined. Open table in a new tab In addition, the corresponding synthetic peptide was made, and its manually identified MS/MS spectrum was used to confirm the assigned sequence of the HRSV ligand. Sequencing of the synthetic peptides was carried out by quadrupole ion trap electrospray MS/MS in a Deca XP LCQ instrument (Thermo Electron) coupled to γLC (Thermo Electron) with a 7–40% acetonitrile gradient for 24 min in the presence of 0.5% acetic acid. An MS/MS mode was used that focused on each hypothetical parental peptide with an isolation width (m/z) of 1.5 Da (36.López D. Calero O. Jiménez M. García-Calvo M. Del Val M. Antigen processing of a short viral antigen by proteasomes.J. Biol. Chem. 2006; 281: 30315-30318Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The charge and mass of the ionic species were determined by high resolution sampling of the mass/charge rank. Collision energy and ion precursor resolution were improved to optimize the fragmentation spectrum. The following synthetic peptides were used as controls in complex stability assays: Flu NP (SRYWAIRTR, HLA-B27-restricted) (37.Wang M. Lamberth K. Harndahl M. Røder G. Stryhn A. Larsen M.V. Nielsen M. Lundegaard C. Tang S.T. Dziegiel M.H. Rosenkvist J. Pedersen A.E. Buus S. Claesson M.H. Lund O. CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening.Vaccine. 2007; 25: 2823-2831Crossref PubMed Scopus (92) Google Scholar) and C4CON (QYDDAVYLK, HLA-Cw4-restricted) (38.Fan Q.R. Garboczi D.N. Winter C.C. Wagtmann N. Long E.O. Wiley D.C. Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-CW4 class I major histocompatibility complex molecule.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7178-7183Crossref PubMed Scopus (78) Google Scholar). RMA-S B27 transfectants, a cell line deficient in TAP that expresses low amounts of MHC class I on the cell surface (27.Villadangos J.A. Galocha B. López de Castro J.A. Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity.J. Immunol. 1994; 152: 2317-2323PubMed Google Scholar), were incubated at 26 °C for 16 h in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. This allows the expression on the cellular membrane of empty MHC class I molecules (without antigenic peptide) that are stable only at 26 °C but not at 37 °C. Later, the cells were washed and incubated for 2 h at 26 °C with various concentrations of peptide in the same medium. Then, the MHC-specific peptides could bind and stabilize the empty MHC class I molecules. Later, the cells were transferred to 37 °C and collected for flow cytometry after 4 h. This allows the internalization of empty MHC class I molecules and thus can discriminate between bound and unbound peptides. MHC expression was measured using 100 γl of a hybridoma culture supernatant containing the ME1 (anti-HLA-B27) monoclonal Ab as described (39.López D. Samino Y. Koszinowski U.H. Del Val M. HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope.J. Immunol. 2001; 167: 4238-4244Crossref PubMed Scopus (21) Google Scholar). Samples were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest Pro 2.0 software (BD Biosciences). Cells incubated without peptide had peak fluorescence intensities close to background staining with secondary Ab alone. The fluorescence index was calculated for each time point as the ratio of peak channel fluorescence of the sample to that of the control incubated without peptide. Binding of peptides was expressed as EC50, which is the molar concentration of the peptide at 50% of the maximum fluorescence obtained at a concentration range of 100–0.001 γm. B27-C1R cells were incubated with the Long strain of HRSV and assayed at different times for the presence of HRSV antigens by flow cytometry. The results indicate that the transfectant cell line incubated with the virus, but not the mock-infected control, expressed HRSV F and G proteins (110 ± 10 mean fluorescence intensity versus 4 ± 3 for the mock control cells). These cells continued to synthesize HRSV viral proteins and secrete infectious virus several months after infection (data not shown). Thus, a B27-C1R transfectant cell line persistently infected with HRSV was obtained in the same manner as previously reported for Epstein-Barr virus-transformed human B-cell lines (40.Bangham C.R. McMichael A.J. Specific human cytotoxic T cells recognize B-cell lines persistently infected with respiratory syncytial virus.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 9183-9187Crossref PubMed Scopus (62) Google Scholar). HLA-bound peptide pools were isolated from large amounts of either healthy or HRSV-infected cells. These peptide mixtures were subsequently separated by reverse-phase HPLC and analyzed by mass spectrometry. Using several software technologies (see “Experimental Procedures”), 209 fragmentation spectra were resolved as peptidic sequences of different human cellular proteins. 2S. Infantes, E. Lorente, E. Barnea, I. Beer, J. J. Cragnolini, R. García, F. Lasala, M. Jiménez, A. Admon, and D. López, manuscript in preparation. Moreover, 10 fragmentation spectra present in the HRSV-infected HLA-bound peptidic pool, but absent in the control uninfected pool (data not shown), were resolved with high confidence parameters as peptides of HRSV viral proteins. Additionally, a human proteome database search failed to reveal the identity of these spectra as human protein fragments, confirming the viral origin of these peptides (data not shown). Two different ion peaks at m/z 409.9 and 614.3 were assigned to the same viral amino acid sequence. These ion peaks corresponded to double (Fig. 1, upper panel) and triple charge (Fig. 1, lower panel) states, respectively, of the peptide HRQDINGKEM, which spans residues 100–109 of the HRSV nucleoprotein (Table I). Virtually all significant fragments of both MS/MS spectra were assigned as daughter ions of the tentative peptidic sequence (Fig. 1). This theoretical assignment was confirmed by identity with the MS/MS spectrum of the corresponding synthetic peptide (supplemental Fig. 1). In addition, the eight other molecular ions were assigned as HLA-restricted viral ligands (Table I and supplemental Figs. 2–9), and their tentative sequences were confirmed as above with the respective synthetic peptide (supplemental Figs. 2–9). Thus, these results indicate that a total of nine HRSV ligands were endogenously processed and presented in the infected cell line. Identification of viral HLA ligands by immunoproteomics analysis is still very limited. A previous study identified 12 viral ligands presented by HLA-A*0201 by differential stable isotope labeling of large amounts of vaccinia virus Ankara-infected cells (41.Meyer V.S. Kastenmuller W. Gasteiger G. Franz-Wachtel M. Lamkemeyer T. Rammensee H.G. Stevanovic S. Sigurdardottir D. Drexler I. Long-term immunity against actual poxviral HLA ligands as identified by differential stable isotope labeling.J. Immunol. 2008; 181: 6371-6383Crossref PubMed Scopus (17) Google Scholar). In another study with a similar approach but 10-fold fewer infected cells, only one HRSV ligand was identified for each HLA-A*0201 or -B*0702 class I molecule (22.Meiring H.D. Soethout E.C. Poelen M.C. Mooibroek D. Hoogerbrugge R. Timmermans H. Boog C.J. Heck A.J. de Jong A.P. van Els C.A. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.Mol. Cell. Proteomics. 2006; 5: 902-913Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In our current report in which the amount of cells used was similar to that in the vaccinia virus study and the peptide pools were compared directly without any labeling, nine endogenously processed HRSV HLA ligands were found. Thus, our current report shows a similar number of HLA ligands between viruses that differ 13-fold in their respective proteome sizes. Therefore, large scale immunoproteomics could allow for the systematic identification of the array of viral HLA ligands. The classical anchor motifs for HLA-B*2705 binding, Arg at position 2 (P2) and basic or aliphatic C-terminal residues (SYFPEITHI database (4.Rammensee H. Bachmann J. Emmerich N.P. Bachor O.A. Stevanoviæ S. SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics. 1999; 50: 213-219Crossref PubMed Scopus (1914) Google Scholar)), were present in all detected viral ligands (Table I). To confirm that HLA-B*2705 is the MHC class I molecule that presents these ligands, MHC-peptide complex stability assays were performed using TAP-deficient RMA-S cells transfected with the HLA-B*2705 molecule. The nine HRSV synthetic peptides induced similar numbers of HLA-peptide surface complexes to a well known HLA-B*2705 epitope from the influenza virus (Fig. 2). In addition, the relative MHC class I affinity was determined for all HRSV peptides. These peptides bound to HLA-B*2705 class I molecules with EC50 values in the range commonly found among natural ligands (Table II). These data indicate that all ligands detected in HRSV-infected cells were endogenously presented in association with the B*2705 molecule.Table IISummary of binding of HLA-B27 ligands of HRSVPeptideaAll peptides were derived from the sequence of the Long strain of HRSV except for Flu NP 383–391 (37), which was used as positive control for HLA-B27 binding.SequenceEC50 ± S.D.bData are means of three to five independent experiments and are expressed as EC50 ± S.D. (see “Experimental Procedures”).γmN 100–109HRQDINGKEM26 ± 5N 184–194RRANNVLKNEM10 ± 1N 195–205KRYKGLLPKDI5 ± 2M 76–84SRSALLAQM9 ± 2M 169–177VRNKDLNTL12 ± 5L 2089–2097GRNEVFSNK18 ± 3M2–22k 150–159KRLPADVLKK9 ± 2P 198–208LRNEESEKMAK14 ± 3NS2 37–45HRFIYLINH11 ± 4Flu NPSRYWAIRTR8 ± 2a All peptides were derived from the sequence of the Long strain of HRSV except for Flu NP 383–391 (37.Wang M. Lamberth K. Harndahl M. Røder G. Stryhn A. Larsen M.V. Nielsen M. Lundegaard C. Tang S.T. Dziegiel M.H. Rosenkvist J. Pedersen A.E. Buus S. Claesson M.H. Lund O. CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening.Vaccine. 2007; 25: 2823-2831Crossref PubMed Scopus (92) Google Scholar), which was used as positive control for HLA-B27 binding.b Data are means of three to five independent experiments and are expressed as EC50 ± S.D. (see “Experimental Procedures”). Open table in a new tab Among the nine HRSV peptides discovered by the proteomics analysis, four (matrix 76–84-, matrix 169–177-, polymerase-, and non-structural protein 2-deri" @default.
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• W2151878384 title "Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells" @default.
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