Multiple, non-conserved, internal viral ligands naturally presented by HLA-B27 in human respiratory syncytial virus-infected cells

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.

Fig. 1.

Identification of the N 100–109 ligand in infected cell extracts by mass spectrometry. MS/MS fragmentation spectra obtained from quadrupole ion trap mass spectrometry of the ion peaks at m/z 614.3 (upper panel) and m/z 409.9 (lower panel) from the HRSV-infected B27-C1R cell extract are shown. The vertical axis represents the relative abundance of the parental ion and each fragmentation ion detected. The horizontal axis corresponds to the m/z region in which significant daughter ions were detected. Ions generated in the fragmentation are detailed, and the sequence deduced from the indicated fragments is shown in the upper right box of each panel.

Fig. 2.

HLA stabilization assay of HRSV synthetic ligands. The stability of HLA-B*2705-peptide complexes on the surface of RMA-S transfectant cells was measured by flow cytometry. The indicated peptides were used at 200 μm. The C4CON (38) and Flu NP (37) peptides were used as negative and positive controls, respectively. The results, calculated as fluorescence index (see “Experimental Procedures”), are the mean (bars) ± S.D. (error bars) of three to four independent experiments.