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 INEBNA-1
G. Stuber et al., 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 INEBNA-1, International immunology, 7(4), 1995, pp. 653-663
B lymphocytes immortalized with EBV in vitro, lymphoblastoid cell line
s (LCL), express eight EBV-encoded proteins, E8V nuclear antigens -1 t
o -6 (EBNA-1 to -6), and latent membrane proteins 1 and 2 (LMP 1 and 2
). After appropriate stimulations of blood lymphocytes from seropositi
ve 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 c
ell surfaces. We posed the question whether this exceptional feature o
f EBNA-1 is due to lack of MHC class I binding peptides. A computer se
arch for 11 human leukocyte antigen (HLA) alleles showed that EBNA-1 h
as a lower number and lower proportion of relevant binding motifs to s
everal alleles than EBNA-2 to -6 and LMP 1 and 2. The low motif number
s in EBNA-1 is in line with its apparent failure to generate a CTL res
ponse, and it may be the consequence of immunoselection allowing the e
xistence of EBV genome-carrying B cells in the immunocompetent hosts.
The binding capacities of synthetic peptides of EBNA-1 and -2 and of t
he immediate early lytic cycle protein BZLF-1 to HLA-A0201 (A2) and HL
A-B7 molecules were tested in an MHC stabilization assay. The peptide
transporter-deficient T2 line, which expresses a low level of HLA-A2 a
nd its HLA-B7 transfectant subline, were used for this purpose because
specifically bound peptides elevate the surface expression of these M
HC 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 B
ZLF-1 peptides were identified as A2 binders, and four EBNA-2 and eigh
t BZLF-1 peptides bound to 87. Thus, we have excluded the possibility
that the inability of the EBNA-1 protein to generate HLA-restricted CT
Ls could be due to the lack of HLA class I binding peptides in its seq
uence. The finding that several EBNA-1 peptides could bind to these tw
o HLA molecules does not, however, necessarily reflect the natural sit
uation because the peptides may not be processed and/or transported to
the cell surfaces. We have stimulated lymphocytes of healthy donors w
ith relevant HLA types with the autologous LCL. The resulting auto-LCL
-reactive culture did not lyse autologous phytohaemagglutinin blasts c
oated with the binder peptides, and we could not generate auto-LCL-spe
cific CTLs by stimulation of lymphocytes with these peptides.