EBV LMP2A affects LMP1-mediated NF-κB signaling and survival of lymphoma cells by regulating TRAF2 expression

Abstract

A mechanism used by Epstein-Barr virus (EBV) for in vitro transformation of B cells into lymphoblastoid cell lines (LCLs) is activation of the NF-B pathway, which is largely mediated by the EBV latent membrane protein 1 (LMP1). LMP1 is coexpressed with LMP2A in many EBV-associated lymphoid malignancies. Since inhibition of NF-B leads to apoptosis of EBV-infected LCLs and lymphoma cell lines, we sought to determine whether LMP1 alone, or in combination with other viral proteins, is responsible for initiating NF-B activation in these cells, thereby playing a role in cell survival. We found that suppression of LMP1 by RNA interference results in inhibition of basal NF-B and induction of apoptosis. Unexpectedly, knockdown of LMP2A also resulted in comparable decrease of NF-B activity and apoptosis. We report that LMP2A protein controls the expression of TRAF2 mRNA, which in turn is necessary for signaling by LMP1. Our data contrast with previous studies showing that transfected LMP1 can signal in the absence of LMP2A or TRAF2, and demonstrate that both LMP2A and TRAF2 are required for survival in naturally infected lymphoma cells and LCLs. These results also support LMP1, LMP2A, and TRAF2 as potential therapeutic targets in a subset of EBV-associated lymphoid malignancies. IntroductionThe association of Epstein-Barr virus (EBV) with several specific lymphoid malignancies is quite consistent, indicating an etiopathogenic role in their development. Support for EBV being an oncogenic virus comes from its ability to infect and transform normal human B cells in vitro, resulting in their immortalization and leading to continuously growing lymphoblastoid cell lines (LCLs). 1 Infection by EBV in healthy individuals is self-limiting, but a distinct sequence of events occurs that allows this virus to establish life-long latency in memory B cells. To achieve this, EBV expresses 2 latent membrane proteins that mimic signals in B cells that are involved in the germinal center reaction and B-cell differentiation into memory B cells. 2 These are the latent membrane protein 1 (LMP1), which functions as a constitutively active CD40 receptor, and LMP2A, containing an immunoreceptor tyrosine-based activation motif (ITAM), thereby mimicking antigen receptor signaling. While these 2 proteins can provide EBVinfected cells with proliferative and antiapoptotic signals, they are also immunogenic, so cells expressing LMP1 and LMP2A are eliminated after acute infection in healthy individuals, and the only surviving EBV-infected B cells that generate the latent reservoir are those that down-regulate these antigenic proteins and differentiate into memory B cells.EBV is thought to lead to the development of lymphoma as a consequence of alterations in the normal viral life cycle, for example as a consequence of impaired immune responses. 3 EBV-associated lymphomas frequently occur in immunodeficient individuals, including those infected with HIV and organ transplant recipients. 4,5 In these lymphomas, tumor cells usually express several EBV-enco...

The Epstein-Barr Virus (EBV)-Induced Tumor Suppressor MicroRNA MiR-34a Is Growth Promoting in EBV-Infected B Cells

“…4d). These data are consistent with previous studies implying a role for LMP2A in LMP1-mediated NF-B activation (18) and our findings that EBV-induced miR-34a expression was NF-B dependent.…”

The Latent Membrane Protein 1 (LMP1)

“…Overexpression of either TRAF was shown to reduce CTAR1-dependent NF-κB activation as well as processing of p100, and dominant-negative TRAF2 inhibited CTAR1 signaling, indicating that both TRAF2 and TRAF3 are involved in the regulation of non-canonical NF-κB by LMP1 (Devergne et al 1996;Kaye et al 1996;Song and Kang 2010). Accordingly, the siRNA-mediated knockdown of TRAF2 caused a marked reduction of total NF-κB activity and the concomitant induction of apoptosis in LCLs (Guasparri et al 2008). However, and in contrast to CD40 signaling, TRAF3 is eliminated by LMP1 through a mechanism independent of the proteasome (Brown et al 2001).…”

c-Myc and Rel/NF-κB Are the Two Master Transcriptional Systems Activated in the Latency III Program of Epstein-Barr Virus-Immortalized B Cells

“…Overactivation of NF-B by LMP1 may also lead to apoptosis by ligandindependent activation of CD95 (31). Consistent with a role in protection against apoptosis, LMP2a has been shown to provide B cells lacking a functional BCR with a survival signal in vivo (8) and to increase NF-B activation (16). LMP2a has also been shown to activate the Notch pathway (2).…”