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.
Highlights
Epstein-Barr virus (EBV) is a B-lymphotropic gammaherpesvirus that establishes latent infection in over 90% of the world’s population [1,2]
We have demonstrated that latent membrane protein 2A (LMP2A) dampens cellular proliferation and activation induced by latent membrane protein 1 (LMP1), which may be due to the LMP2A-associated decrease in the levels of TRAF2, a signaling protein used by LMP1
LMP2A allows B cells carrying LMP1 to enter the germinal center during an immune response, a site that gives rise to EBVassociated tumors in humans
Summary
Epstein-Barr virus (EBV) is a B-lymphotropic gammaherpesvirus that establishes latent infection in over 90% of the world’s population [1,2]. EBV may persist for the life of the host in resting memory B cells where a limited number of viral genes are expressed [3]. EBV has the unique ability of transforming primary human B cells into lymphoblastoid cell lines (LCLs) expressing the latency III program of gene products [4], including six EBV nuclear antigens (EBNA1, -2, -3A, -3B, -3C and LP) as well as three latent membrane proteins (LMP1, -2A, and 2B), and multiple non-coding RNAs (EBERs and miRNAs). LMP1 signaling plays a role in B cell survival by upregulating Bcl-2, A20 and Mcl-1 in human B cell lines and murine transgenic lymphomas [6,11,12,13]. LMP1 has been shown to block germinal center formation [10,16], and to synergize with CD40 signaling to enhance proliferation and immunoglobulin production [16]
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