HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA-1, EBNA-2 and BZLF-1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1.
B lymphocytes immortalized with EBV in vitro, lymphoblastoid cell lines (LCL), express eight EBV-encoded proteins, EBV nuclear antigens -1 to -6 (EBNA-1 to -6), and latent membrane proteins 1 and 2 (LMP 1 and 2). After appropriate stimulations of blood lymphocytes from seropositive individuals, MHC-restricted cytotoxic T cells (CTL), which lyse LCL cells, can be generated in vitro. Such CTLs can recognize EBNA-2 to -6, and LMP 1 and 2, but not EBNA-1-derived peptides presented on the cell surfaces. We posed the question whether this exceptional feature of EBNA-1 is due to lack of MHC class I binding peptides. A computer search for 11 human leukocyte antigen (HLA) alleles showed that EBNA-1 has a lower number and lower proportion of relevant binding motifs to several alleles than EBNA-2 to -6 and LMP 1 and 2. The low motif numbers in EBNA-1 is in line with its apparent failure to generate a CTL response, and it may be the consequence of immunoselection allowing the existence of EBV genome-carrying B cells in the immunocompetent hosts. The binding capacities of synthetic peptides of EBNA-1 and -2 and of the immediate early lytic cycle protein BZLF-1 to HLA-A0201 (A2) and HLA-B7 molecules were tested in an MHC stabilization assay. The peptide transporter-deficient T2 line, which expresses a low level of HLA-A2 and its HLA-B7 transfectant subline, were used for this purpose because specifically bound peptides elevate the surface expression of these MHC molecules. Of five synthetic nonamer EBNA-1 peptides which include the relevant binding motif, four bound to A2. In a series of 20-amino acid-long overlapping EBNA-1 peptides none showed binding to A2, while eight peptides bound to B7. Two 20-amino acid-long EBNA-2 and seven BZLF-1 peptides were identified as A2 binders, and four EBNA-2 and eight BZLF-1 peptides bound to B7. Thus, we have exclude the possibility that the inability of the EBNA-1 protein to generate HLA-restricted CTLs could be due to the lack of HLA class I binding peptides in its sequence. The finding that several EBNA-1 peptides could bind to these two HLa molecules does not, however, necessarily reflect the natural situation because the peptides may not be processed and/or transported to the cell surfaces. We have stimulated lymphocytes of healthy donors with relevant HLA types with the autologous LCL.(ABSTRACT TRUNCATED AT 400 WORDS)
Stuber
G
G
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden.
Dillner
J
J
Modrow
S
S
Wolf
H
H
Székely
L
L
Klein
G
G
Klein
E
E
D000483:Alleles; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens; D015234:HLA-A Antigens / Q000378:metabolism*; D015793:HLA-B7 Antigen / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*
D000956:Antigens, Viral; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D015234:HLA-A Antigens; D015793:HLA-B7 Antigen; D015534:Trans-Activators; D014764:Viral Proteins