Epstein-Barr Virus LMP2A-Induced B-Cell Survival in Two Unique Classes of EμLMP2A Transgenic Mice

Epstein-Barr virus (EBV) is able to infect primary human B cells in culture, transforming them into lymphoblastoid cell lines (LCLs). Upon infection, the virus enters a latent life cycle characterized by the expression of six EBV nuclear antigens (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP) and three latently expressed membrane proteins (LMP1, LMP2A, and LMP2B) (17). These latent viral proteins dramatically alter in vitro B-cell biology as characterized by upregulated expression of activation and adhesion markers (CD23, CD39, CD40, CD44, LFA-1, LFA-3, and ICAM-1), decreased dependence on high-serum medium, and secretion of immunoglobulin (17). However, EBV latency in defined culture systems in vitro is quite different from in vivo latency. In latently infected individuals, virus can be isolated from immunoglobulin M-positive (IgM+) IgD− resting memory B cells which have lost surface expression of the costimulatory molecule B7-1 and the activation marker CD23 (1, 34, 35). Of the nine latent gene products detected in vitro, only EBNA1 and LMP2A transcripts are reproducibly detected in latently infected human B cells (6, 34, 40, 46). Although latent virus has been isolated from peripheral B cells in otherwise healthy individuals (1, 6, 8, 15, 34, 35, 40, 46), latently infected B cells may reside in bone marrow or other lymphatic sites producing latently infected progeny cells which account for the stable number of infected peripheral B cells in otherwise healthy individuals (21, 23, 35, 50, 51). Considerable genetic and biochemical research has elucidated many aspects of LMP2A biology in vitro. LMP2A has 12 transmembrane domains and is expressed in patches on the surface of latently infected cells (24, 25, 42). The Lyn protein tyrosine kinase (PTK) binds to LMP2A via tyrosine residue 112 and is essential for the constitutive LMP2 phosphorylation detected in LCLs (12). Lyn-dependent phosphorylation allows other PTKs to bind specific sites within the LMP2A amino terminal cytoplasmic tail (10, 12). The Syk PTK specifically binds to the LMP2A ITAM domain at tyrosine residues 74 and 85 (11). Syk bound to LMP2A becomes constitutively phosphorylated and is unable to participate in B-cell receptor (BCR)-initiated signal transduction (11). Through interactions with these and other cellular proteins, LMP2A is able to downmodulate intracellular signaling cascades mediated by the BCR, the CD19 complex, and major histocompatibility complex class II (31, 33, 38). Although expressed in Hodgkin's disease and nasopharyngeal carcinoma cells (2, 4, 7, 36, 37), LMP2A is not essential for EBV transformation in vitro (27–29). Transgenic mice expressing EBV latency proteins under transcriptional control of an immunoglobulin promoter have provided a convenient system for analyzing latent viral gene function in B cells in vivo. LMP1 transgenic mice develop lymphomas which exhibit upregulated expression of the antiapoptotic proteins Bcl-2 and A20, as well as the Myc oncoprotein (19). The episomal maintenance protein EBNA1 is able to induce monoclonal B-cell lymphomas in EμEBNA1 transgenic mice (48, 49). These results are surprising in that EBNA1 has no transforming ability in vitro. Recently described research with EμLMP2A transgenic mice suggest a role for LMP2A in B-cell survival in vivo (5). LMP2A transgene expression alters early B-cell development in the bone marrow during a transition requiring preBCR activation of Syk PTK. LMP2A expression blocks immunoglobulin heavy-chain expression while allowing subsequent immunoglobulin light-chain rearrangement. The resulting peripheral B cells do not express a BCR complex and yet, surprisingly, survive in the spleen without BCR stimulation. LMP2A is also able to drive B-cell development when EμLMP2A transgenic animals are crossed into a recombinase-activating gene (RAG) null background. These results indicate that LMP2A may be able to provide a yet-uncharacterized proliferative and survival signal to B cells in vivo. Three additional EμLMP2A transgenic mice have been isolated which have relatively normal proportions of BCR-positive B cells in a genetically wild-type background. However, when crossed into a RAG null background, LMP2A transgene expression in these new EμLMP2A animals is also able to provide a survival signal to BCR-negative peripheral B cells. As-yet-undefined epigenetic effects modulating either the time or level of LMP2A expression may be controlling the EμLMP2A transgenic phenotype in a wild-type genetic background.