An Epstein-Barr virus-encoded protein, LMP2, blocks the effects of surface immunoglobulin (slg) cross-linking on calcium mobilization and on lytic reactivation of EBV in latently infected and growth-transformed primary human B lymphocytes. In wild-type EBV-transformed cells, LMP2 is constitutively tyrosine phosphorylated and is associated with Lyn and Syk protein-tyrosine kinases (PTKs). Baseline Lyn PTK activity is substantially reduced, and slg cross-linking fails to activate Lyn, Syk, Pl3-K, PLC gamma 2, Vav, Shc, and MAPK. Syk, Pl3-K, PLC gamma 2, and Vav are constitutively tyrosine phosphorylated, and their tyrosine phosphorylation does not change following slg cross-linking. In contrast, cross-linking slg on cells transformed by LMP2 null mutant EBV recombinants triggers the same protein tyrosine kinase cascade as in noninfected B lymphocytes. These data are consistent with a model in which LMP2 is a constitutive dominant negative modulator of slg receptor signaling through its effects on Lyn, Syk, or regulators of these kinases.

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-ype control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.Epstein-Barr virus (EBV) is a herpes virus that establishes latency in B lymphocytes during primary infection. Latent infection of B lymphocytes is likely to be essential for perpetuation of this virus in human populations, since virus reactivation from latency is the major reservoir of virus for infection of susceptible hosts. In latent infection, the virus can express six nuclear proteins [EBV nuclear antigens (EBNAs)], two integral membrane proteins [latent membrane proteins (LMPs)], and two small RNAs, thereby causing proliferation of the infected lymphocyte (1-3). Latently infected lymphocytes can cause a lymphoproliferative disease in immunodeficient individuals (4-6). The EBNAs and LMPs are being intensively studied since they are likely to be involved in the maintenance or regulation of latency or of the concomitant effects on cell proliferation. This report focuses on LMP2, the protein(s) most recently discovered to be expressed in latently infected B lymphocytes (7-9).Two forms of LMP2 (designated LMP2A and -2B) are expressed in latently infected B lymphocytes. Both forms are encoded by highly related mRNAs composed of eight exons with a single long open reading frame, differing only in their first exons (10-12). The first exon of LMP2A encodes a 119-amino acid hydrophilic N terminus that is unique to LMP2A. The promoter for LMP2B is in the first LMP2A intron, and the first LMP2B exon is noncoding. The first codon of the second exon in LMP2A and -2B is a methionine codon, at which translation of LMP2B initiates. That methionine is followed by 12 predicted hydrophobic transmembrane domains separated by short reverse turns and a 27-

The Epstein-Barr virus (EBV) nuclear protein 3C (EBNA 3C) is essential for EBV-mediated transformation of primary B lymphocytes, is turned on by EBNA 2, and regulates transcription of some of the viral and cellular genes which are regulated by EBNA 2. EBNA 2 is targeted to response elements by binding to the DNA sequence-specific, transcriptional repressor protein J. We now show that EBNA 3C also binds to J. EBNA 3C causes J to not bind DNA or EBNA 2. J DNA binding activity in EBV-transformed lymphoblastoid cells is consequently reduced. More than 10% of the EBNA 3C coimmunoprecipitated with J from extracts of non-EBV-infected B lymphoblasts that had been stably converted to EBNA 3C expression. EBNA 3C in nuclear extracts from these cells (or in vitro-translated EBNA 3C) prevented J from interacting with a high-affinity DNA binding site. Under conditions of transient overexpression in B lymphoblasts, EBNA 2 and EBNA 3C associated with J and less EBNA 2 associated with J when EBNA 3C was coexpressed in the same cell. EBNA 3C had no effect on the activity of a ؊512/؉40 LMP1 promoter-CAT reporter construct that has two upstream J sites, but it did inhibit EBNA 2 transactivation of this promoter. These data are compatible with a role for EBNA 3C as a ''feedback'' down modulator of EBNA 2-mediated transactivation. EBNA 3C could, in theory, also activate transcription by inhibiting the interaction of the J repressor with its cognate DNA. The interaction of two viral transcriptional regulators with the same cell protein may reflect an unusually high level of complexity or stringency in target gene regulation.

No abstract

Using second-site homologous recombination, Epstein-Barr virus (EBV) recombinants were constructed which carry an LMP2A mutation terminating translation at codon 19. Despite the absence of LMP2A or LMP2A cross-reactive protein, the recombinants were able to initiate and maintain primary B-lymphocyte growth transformation in vitro. EBNA1, EBNA2, and LMP1 expression was unaffected by the LMP2A mutation. The LMP2A mutant recombinant EBV-infected lymphoblastoid cell lines (LCLs) were identical to wild-type recombinant EBV-infected control LCLs with respect to initial outgrowth, subsequent growth, sensitivity to limiting cell dilution, sensitivity to low serum, and growth in soft agarose. The permissivity of LCLs for lytic EBV infection and virus replication was also unaffected by the LMP2A mutation.