Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein whichhas transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-κB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to β-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxyterminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells crosslinking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.

The Epstein‐Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B‐cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis‐acting element, various TP1 promoter‐reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B‐cell line BL41‐P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between −258 and −177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein‐DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel‐shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis‐element of the TP1 promoter.

Epstein‐Barr virus (EBV) infects human primary B lymphocytes and induces and maintains proliferation of these cells efficiently in vitro. Mutants of Epstein‐Barr virus which express EBV nuclear antigen 2 (EBNA2) in a conditional fashion allow dissection of individual contributions of viral genes to B cell immortalization. EBNA2 is a transcriptional activator of cellular and viral genes, including the viral latent membrane protein 1 (LMP1), which is essential for B cell immortalization and has oncogenic effects in non‐lymphoid cells. To analyze the role of this gene in B cell immortalization, LMP1 was constitutively expressed in B cells infected with EBV carrying a conditional EBNA2 allele. In the absence of functional EBNA2, LMP1 was incapable of sustaining B cell proliferation in two independent assays but induced a phenotype consistent with prolonged cell viability. Activation of CD40 displayed a comparable phenotype. These data indicate that both CD40 activation and LMP1 expression may use a common pathway for B cell activation. Proliferation of human B cells, however, requires one or more additional signals triggered by EBNA2.

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Epstein-Barr virus (EBV), a human herpesvirus, is associated with several human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, T-cell lymphoma, gastric carcinoma, Hodgkin lymphoma, and immunoblastic lymphoma in immunocompromised individuals. The virus has the ability to immortalize primary B cells in vitro. In these immortalized lymphoblastoid cell lines, only a few viral genes are expressed, coding for six nuclear proteins (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP) and three latent membrane proteins (LMP1, LMP2A, and LMP2B) (for a review, see reference 35). Five of the nuclear antigens (EBNA1, -2, -3A, and -3C seem to be absolutely required for B-cell immortalization (10,23,32,68), whereas EBNA-LP and LMP2A seem to affect the efficiency of the process (6,23,47).EBNA2 plays a pivotal role in B-cell immortalization, since a natural occurring EBV mutant, the P3HR1 strain, carrying a deletion of the EBNA2 gene, has lost the ability to transform primary B cells. Reintroduction of the EBNA2 gene in the viral genome by homologous recombination or complementation can restore the immortalizing capacity of the virus (23). EBNA2 contributes to B-cell immortalization most likely by its ability to act as transcriptional modulator of cellular and viral gene expression. It activates the transcription of the B-cell activation markers CD21 and CD23 (9,11,70) and the tyrosine kinase c-Fgr (37) and downregulates the expression of the immunoglobulin heavy-chain locus (Ig) (30). In addition, EBNA2 transactivates the viral promoters of the three latent membrane proteins LMP1, LMP2A, and LMP2B and the Cp promoter, which regulates the transcription of the EBNA genes (17,64,75,76). EBNA2 does not bind to DNA directly (45, 77) but is recruited to EBNA2-responsive elements by interacting with the transcriptional factors 25,69,78) and PU