Epstein-Barr Virus LMP2A Reduces Hyperactivation Induced by LMP1 to Restore Normal B Cell Phenotype in Transgenic Mice

Abstract

Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors.

Mouse model for acute Epstein–Barr virus infection

“…Here LMP2A appeared to negatively modulate the function of LMP1, rescuing the loss of GC B cells caused by LMP1 expression. A clear difference with respect to our previous and present work is the presence of large numbers of LMP-expressing cells in the mice analyzed by Longnecker and colleagues (32), suggesting that the levels of LMP1 expression were insufficient to induce T-cell immune surveillance as it is seen in the present mouse model and in human EBV infection. Indeed, there is evidence that LMP2A augments LMP1 signaling in human EBVinfected cells (33).…”

Evasion of affinity-based selection in germinal centers by Epstein–Barr virus LMP2A

“…Consistent with this observation, LMP2A coexpression reversed LMP1-induced B-cell hyperactivation phenotypes in a murine model (22). Pathways suppressed by LMP2A expression included apoptosis, Toll-like receptor signaling, B-cell receptor signaling, and the TNFα/ NF-κB pathway (Table S3).…”

“…However, B-lineage-specific expression of LMP1 inhibits GC formation (20) or causes B-cell ablation through immune surveillance (21). LMP2A does not affect affinity maturation of B cells in GCs when expressed in B cells of a transgenic mouse line (22). Constitutive expression in transgenic mice used in these previous studies may be inappropriate for studying the influence of infection-induced virus proteins on B-cell differentiation, particularly in GCs.…”

Epstein-Barr Virus LMP1-Mediated Oncogenicity

“…In classic studies, LMP1 was found to transform rodent fibroblasts, to drive epithelial xenograft tumor formation (7,66), and to accelerate B-cell lymphomagenesis (9). When expressed alone or together with LMP2A from an early stage of B-cell development, immune surveillance prevents lymphomagenesis (8,67). Notably, the loss of T-cell surveillance results in LMP1-driven fatal lymphoproliferative disease within 60 days (8).…”