Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins

Abstract

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gtll-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-13-galactosidase. Serum of a rabbit immunized with BFRF3-13galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-1-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens. Epstein-Barr virus (EBV) is a ubiquitous member of the human herpesvirus family and is associated with a still increasing number of disease syndromes. EBV-specific diagnosis is based on the combination of different serological parameters. These parameters include the detection of antibodies of different classes against early antigens (EA), Epstein-Barr nuclear antigens (EBNA), viral capsid antigens (VCA), and membrane antigens. Polypeptides belonging to the EBNA and EA complexes and their role in immunodiagnosis has been studied extensively (24, 29, 39). In contrast, only few studies have addressed in detail the human immune response to viral structural polypeptides. Studies of the structural polypeptides are hampered by the molecular complexity of the VCA complex, the polyspecificity of the human sera used to detect them, and the lack of an efficient virus production system. EBV virions may consist of some 30 polypeptides (9). Localization within the virion and a role in penetration, assembly, and budding have been suggested for only a few. The viral capsid may be composed of at least seven proteins (7), of which the major capsid protein encoded by open reading frame (ORF) BcLF1 (37) and a protein of 36 kDa encoded by BGLF2 (5, 33) have been described in detail. Another VCA protein, gpllO, is encoded within the BALF4 reading frame (34). This protein remains associated with cellular cytoplasmic and nuclear membranes and is not associated with the virion (15). Recently, we have identified two new immunologically dominant VCA proteins with molecular sizes of 18 and 40 kDa (36). These two proteins are structurally associated with the viral capsid and are recognized by antibodies from almost all EBV carriers.

Two 21-kilodalton components of the Epstein-Barr virus capsid antigen complex and their relationship to ZEBRA-associated protein p21 (ZAP21)

“…The BLRF2 protein had not been previously characterized. Another candidate was the recently described BFRF3 component of VCA (25), which had been previously misclassified as an early protein (7). In this study, we demonstrate that BLRF2 is a component of the late p21 complex and we investigate the relationship of BFRF3 and BLRF2 to ZAP21.…”

“…1B and data not shown). Both proteins are basic and are expressed from late transcripts (2,3,25) (Fig. 2).…”

“…SJ is a human serum from a healthy EBV-positive donor. Anti-BFRF3 is a rabbit serum raised against a ␤-galactosidase-BFRF3 fusion protein (25). Anti-BLRF2 is a rabbit serum raised against a TrpE-BLRF2 fusion protein by methods previously described (22).…”

“…BALF4 is thought to be a major immunogen of VCA (13). BdRF1 encodes a 40-kDa protein which was recently characterized as a diagnostically relevant marker antigen (23,25). BFRF3 encodes an 18-to 21-kDa protein which is another immunodominant marker for EBV infection (23)(24)(25).…”

Native early antigen of Epstein–Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

“…Monoclonal and monospecific polyclonal antisera were produced in mice and rabbits using synthetic peptides or purified recombinant protein, as described elsewhere. Antibodies to characterize EBV proteins consisted of OT13B (anti-EA-p138, BALF2) [Zeng et al, 1997], rabbit anti-EBNA1 (BKRF1) [Meij et al, 2000], rabbit anti-DNase (BGLF5) [Stolzenberg et al, 1996;Rowe et al, 2007], OT14E (anti-EA-p47, BMRF1) [Zhang et al, 1998], BZ-1 (anti-Zebra, BZLF1) [Rowe et al, 1988], OT41A (anti-VCA-p40, BdRF1) [van Grunsven et al, 1993b], and OT15E (anti-VCA-p18, BFRF3) [van Grunsven et al, 1994].…”