Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Abstract

The recognition of short viral peptides associated with human histocompatibility complex (human leukocyte antigen (HLA) 1 ) class I molecules on the cell surface allows cytotoxic T lymphocytes (CTLs) to recognize and kill virus-infected cells (1). 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). 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 ␤ 2 -microglobulin. Usually, two major anchor residues in the antigenic peptide, at position 2 and the C terminus (3, 4), must be deeply accommodated into specific pockets of the antigen recognition site of the HLA class I molecule to stabilize the nascent complexes (5, 6) and allow for their subsequent transport to the cell membrane where they are exposed for CTL recognition (7).Human respiratory syncytial virus (HRSV) (8), a member of the Paramyxoviridae family, is the single most important cause of bronchiolitis and pneumonia in infants and young children (9 -11). 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, 13) and in the elderly (14, 15). 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 ϩ T lymphocytes are required for the clearance of virus-infected cells (16). Previously, several HRSV epitopes restricted by different HLA class I molecules were identified using CTLs from seropositive individuals (17-21). 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). 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). 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-B27From the ‡Unidad

Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus

“…And lastly, the analysis of the immunoprevalence and immunodominance of the HLA class II response described in this report together with the HLA class I response from previous immunoproteomics studies (12,13,26) could have implications for vaccine development. The 3Ј core and intermediate genes translate 52% of the HRSV proteome; however, they include 94% and 100% of natural HLA class I and II , N (nucleoprotein), P (phosphoprotein), M (matrix protein), SH (small hydrophobic protein), G (glycoprotein), F (fusion protein), M2 (matrix protein 2), M2-2 (matrix protein 22k), and L (polymerase).…”

“…5). In previous immunoproteomics studies (12,13,26), analyses of the HLA class I-restricted antiviral immune response against HRSV identified 16 natural viral ligands by mass spectrometry. This analysis showed that 12 of the HRSV ligands that were detected were included in proteins encoded by the 3Ј group, whereas only three and one of them were integrated into proteins that were encoded by the intermediate and 5Ј groups, respectively (Fig.…”

“…The peaks were identified using the Proteome Discoverer 1.0 SP1 (Thermo-Fisher) and Bioworks Browser 3.3.1 SP1 (Thermo-Fisher) software programs (17) using the human and virus parts of the NCBI database (Jan 2014), which included 776,554 proteins. The search was not limited by enzymatic specificity; the peptide mass tolerance was set to 0.005 Da, and the fragment ion tolerance was set to 0.5 Da (12,13,18). This search was not limited by any methodological bias (e.g.…”

“…HRSV Infection of the Human JY Epstein-Barr-Transformed Cell Line-JY cells (HLA-DRB1*0404, -DRB4*0101, -DRB1*1301, and -DRB3*0101 or -DRB3*0201) were incubated with the HRSV Long strain and assayed at different time points for the presence of HRSV antigens using flow cytometry, as previously described, with either Epstein-Barr-transformed human B-cell lines (11) or other cell lines (12), to obtain a persistently infected JY-cell line that synthesized HRSV viral proteins and secreted infectious virus several months after infection (13).…”

“…individual protein selection or HLA consensus scoring algorithm use). The identified peptides were selected if the following criteria were met: Sequest Xcorr Ͼ1.5 for singly, Ͼ2.5 for doubly, Ͼ3.2 for triply, and Ͼ4.3 for quadruply charged peptides, as well as a mass accuracy of 0.005 Da (12,13,18). The following variable modifications was also analyzed: cysteinilation (C), oxidation (M, P, H, F, Y, and W), methylation (C, and N terminus), and phosphorylation (Y, S, and T).…”