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

Susana Infantes,Elena Lorente,Eilon Barnea,Ilan Beer,Juan José Cragnolini,Ruth García,Fátima Lasala,Mercedes Jiménez,Arie Admon,Daniel López

doi:10.1074/mcp.m900508-mcp200

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.

Highlights

Cytotoxic T lymphocyte (CTL)-mediated death of virusinfected 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 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]
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]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Antibodies (Abs)—B27-C1R is a transfectant [23] of the human lymphoid cell line HMy2.C1R (C1R) that expresses its endogenous HLA class I antigens at a low level [24, 25]. RMA-S B27 transfectants, a cell line deficient in TAP that expresses low amounts of MHC class I on the cell surface [27], 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. 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 ␮M

RESULTS AND DISCUSSION

Experimental massa

Percentage of proteomee