Nuclear antigen 1 (EBNA‐1) is one of the key functions of the oncogenic DNA virus, Epstein‐Barr virus (EBV), and is the only viral protein consistently expressed in EBV‐associated malignancies. EBNA‐1 binds in a site‐specific manner to the viral DNA and is essential for viral replication, as well as for maintaining the genome as an extrachromosomal episome within infected cells. EBNA‐1 is not recognized by the cellular immune system. Here we demonstrate that, in addition to its known DNA binding properties, EBNA‐1 can also act as a strong RNA binding protein, interacting with diverse substrates in vitro, including the EBV‐encoded RNA polymerase III transcript EBER1 and the HIV‐encoded transactivation response (TAR) element. We also show that EBNA‐1 can bind exon sequences derived from its own RNA expressed from the Fp promoter, as found in Burkitt's lymphoma‐related cells and in nasopharyngeal carcinomas. EBNA‐1 has been identified as a component in an RNA complex; moreover, an anti‐EBNA‐1 antibody 1H4‐1, that does not inhibit DNA binding, blocks binding to RNA. Arginine/glycine‐containing (so‐called ‘RGG’) motifs have been found in an increasing number of proteins that interact with RNA. The EBV antigen contains three potential ‘RGG’ motifs located around an internal glycine/alanine‐rich repetitive sequence in the protein, and outside the region of EBNA‐1 mapped previously as essential for viral DNA replication and other functionally defined properties. These motifs could be involved in the observed binding between EBNA‐1 and RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies directed against the Epstein-Barr virus nuclear protein 1 (EBNA1) were used to examine conventional paraffin sections from a series of EBV-associated lymphoproliferative disorders by immunohistochemistry. The presence of latent EBV infection in tumor cells was determined by in situ hybridization for the Epstein-Barr virus early RNAs (EBERs). Of those EBER-positive cases a total of 28 of 40 cases of Hodgkin's disease, 3 of 3 cases of Burkitt's lymphoma, and 8 of 8 cases of human immunodeficiency virus-associated cerebral B-cell lymphoma expressed detectable amounts of EBNA1. In the positive cases, expression was confined to the tumor cells. No reactivity was detected in EBV-negative cases of the above tumors or in 8 cases of EBV-negative cases of large cell anaplastic non-Hodgkin lymphoma. This report provides the first unequivocal evidence for the expression of the EBNA1 protein in the tumor cells of Hodgkin's disease and validates an important reagent with which to analyze the role of EBV in various virus-associated malignancies.

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.

We have previously developed two monoclonal antibodies against the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1), designated 1H4 and 2B4. Both detect EBNA1 by in situ staining in established EBV-positive tumours, e.g. Hodgkin's lymphoma and nasopharyngeal carcinoma. An association of EBV with other tumours, notably breast carcinomas, has been reported but remains controversial. Using the antibody 2B4, a nuclear protein has been detected in breast carcinomas that were EBV-negative by other methods, suggesting cross-reactivity with a cellular protein. Furthermore, an association of EBV with various other carcinomas has been reported on the basis of 2B4 immunohistochemistry. Here we show that 2B4 also binds to MAGE-4, a cancer testis antigen expressed in a variety of tumour cells, including breast carcinoma, seminoma and EBV-negative cases of Hodgkin's lymphoma. We conclude that the 2B4 antibody is not suitable for the detection of EBV infection but that additional techniques, particularly in situ hybridization for the detection of the EBV-encoded RNAs (EBERs), should be employed to confirm the presence of EBV. Our results add to the evidence indicating that breast cancer is not an EBV-associated disease.