Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.

Abstract

Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) Epstein-Barr virus (EBV) converts normal B lymphocytes into immortalized lymphoblastoid cell lines (1, 2). Conversion is regularly accompanied by the appearance of the EBV-determined nuclear antigen (EBNA) (3). EBNA has been detected in all EBV-genome-carrying cells by anticomplement immunofluorescence (ACIF) (4), including the two EBV-carrying human tumors, Burkitt lymphoma and nasopharyngeal carcinoma (5, 6). EBNA appears at a very early state of the primary transforming interaction between the virus and the B lymphocytes already before the onset of cellular DNA replication (7). In mitotic cells EBNA remains associated with the metaphase chromosomes (4), in contrast to the papovavirus T antigens that spread to the cytoplasm. This is in line with the strong DNA-binding properties of EBNA (8).The biochemical properties of EBNA have been studied in several laboratories. Proteins of various size classes varying from 200 to 48 kDa have been shown to react with human anti-EBNA-positive antisera (9-12). Strnad et al. showed that extracts of proteins of molecular size between 63 and 67 kDa were capable of inhibiting the ACIF reaction, and a correlation with an EBV-specific antigen varying in size between different EBV-carrying cell lines was suggested (12). Recent transfection studies with EBV DNA fragments have indicated that a nuclear antigen can be induced by BamHI K fragment (13). The BamHI K fragment contains the third internal repeat region (IR3). It consists of repeated hexa-or nonanucleotide sequences of only three nucleotide triplets: GGG, GCA, and GGA (14). Multiple homologous sequences are present on the human chromosomes, suggesting that the virus may originally have picked up this sequence from the host cell genome (15).Different isolates of EBV contain IR3 sequences of varying length (14). The length of the IR3 sequence is directly correlated with the size of an EBV-associated nuclear antigen varying in size between 68 and 85 kDa (16).The reading frame for transcription of this protein has recently been determined by immunizing rabbits with fusion proteins made in bacteria carrying plasmid clones containing P-galactosidase and IR3, joined in all three reading frames. The protein corresponding to reading frame 2 induced antibodies in rabbits that reacted with the IR3-related proteins on immunoblotting (17). However, EBNA reactivity of these antibodies was not proven by an ACIF reaction.If the IR3 region codes for EBNA or one of its major components, it should be possible to induce EBNA-specific, ACIF-reactive antibodies, by immunizing rabbits wit...

Deiminated Epstein‐Barr virus nuclear antigen 1 is a target of anti–citrullinated protein antibodies in rheumatoid arthritis

“…It has an unusual structure in that it contains in its central portion a Gly-Ala repeat that constitutes one-third of the molecule (30). This repeat represents a dominant epitope in the anti-EBNA-1 immune response that follows EBV infection (31,32), but antibodies against other portions of the molecule are present in immune sera. Healthy individuals, as well as those affected by autoimmune or EBVrelated disorders, produce antibodies to the N-terminal EBNA-1 sequence that contains the 6-fold Gly-Arg repeat (28).…”

Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma.

“…Total cell extracts were separated in SDS/7.5% polyacrylamide gels, blotted on nitrocellulose filters, and probed with previously characterized human sera PG or HR containing high antibody titers to EBNA-1 to -6. Monospecific reagents included affinity purified anti-107 human antibodies and the absorbed human serum JAC specific for EBNA-1 (22), the monoclonal antibody (mAb) PE-2, which detects both the EBNA-2A and -2B isotypes (23), the mAbs JF186 (24) and S-12 (25), which are specific for EBNA-5 and LMP-1, respectively. EBNAs and LMP-1 staining were performed by indirect immunofluorescence with fluorescein-conjugated rabbit anti-human and rabbit anti-mouse IgG, respectively.…”

Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides

“…A control antigen, the EBV EBNA-l-derived peptide whose antigenicity is already well established (Dillner et al, 1984), was included in the synthesis and displayed strong reactivity with anti-EBVpositive human sera (Fig. 1).…”

“…The sequence of HCMV strain AD169 ppl50 (Jahn et al, 1987) was divided into 95 20 amino acid stretches with a nine amino acid overlap between adjacent peptides. These overlapping peptides and a peptide derived from the EpsteinBarr virus (EBV) EBNA-I sequence, known to be highly reactive with EBV-positive sera (Dillner et al, 1984), were synthesized by modified solid-phase synthesis on chemically activated polyethylene pins using the Fmoc/t-Bu protection strategy (Geysen et al, 1987 b). Monitoring of condensation was performed using bromophenol blue (Krchfifik & V~gner, 1990).…”