Autoactivation of the Epstein-Barr virus oncogenic protein LMP1 during type II latency through opposite roles of the NF-kappaB and JNK signaling pathways

Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-kappaB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Mechanisms of EBV-mediated oncogenesis

Epstein-Barr virus (EBV) is the DNA tumor virus, which is known to be relevant to various cancers. EBV maintains latent infection in cancer cells, and there are three types of latent infection (type I-III) according to the patterns of viral latent genes expression. EBV has the ability to transform B cells into immortalized lymphoblastoid cell lines (LCL) showing type III latency, in which all latent genes are expressed. The mechanism of B-cell transformation has provided a model of EBV-associated lymphomas in immunosuppressed individuals. In type I and II latency, the limited numbers of latent genes are expressed. Previous studies have demonstrated the oncogenic functions of latent EBV genes including nuclear antigen EBNA1, membrane protein LMP1 and LMP2A. In addition, we have demonstrated that EBV-encoded small RNA EBERs play a significant role in oncogenesis. Here we summarize recent progresses in the studies on molecular mechanisms of EBV-mediated oncogenesis.     

TRADD domain of Epstein-Barr virus transforming protein LMP1 is essential for inducing immortalization and suppressing senescence of primary rodent fibroblasts

Mutation analysis of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-induced B-cell immortalization revealed two transformation effector sites, TES1 and TES2. TES2 mediates the interaction with tumor necrosis factor receptor-associated death domain protein (TRADD) and plays a key role in transactivating NF-kappa B and AP-1. Recombinant EBV containing LMP1 with TES2 deleted induces a limited proliferation of B cells. The present study shows that a mutant with an LMP1 site-specific mutation at TES2, LMP1(TRADD), initially stimulates cell growth and significantly extends the life span of MEF. However, it is not sufficient for the immortalization of MEF, and MEF-LMP1(TRADD) cells eventually enter growth arrest. Further analysis reveals that although LMP1(TRADD) promotes cell growth, it does not prevent the eventual onset of senescence and the expression of tumor suppressor p16(Ink4a).     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.     

The signaling pathways of Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) in latency and cancer

Epstein-Barr virus (EBV) is a ubiquitous virus with infections commonly resulting in a latency carrier state. Although the exact role of EBV in cancer pathogenesis remains not entirely clear, it is highly probable that it causes several lymphoid and epithelial malignancies, such as Hodgkin’s lymphoma, NK-T cell lymphoma, Burkitt’s lymphoma, and nasopharyngeal carcinoma. EBV-associated malignancies are associated with a latent form of infection, and several of these EBV-encoded latent proteins are known to mediate cellular transformation. These include six nuclear antigens and three latent membrane proteins. Studies have shown that EBV displays distinct patterns of viral latent gene expression in these lymphoid and epithelial tumors. The constant expression of latent membrane protein 2A (LMP2A) at the RNA level in both primary and metastatic tumors suggests that this protein might be a driving factor in the tumorigenesis of EBV-associated malignancies. LMP2A may cooperate with the aberrant host genome, and thereby contribute to malignant transformation by intervening in signaling pathways at multiple points, especially in the cell cycle and apoptotic pathway. This review summarizes the role of EBV-encoded LMP2A in EBV-associated viral latency and cancers. We will focus our discussions on the molecular interactions of each of the conserved motifs in LMP2A, and their involvement in various signaling pathways, namely the B-cell receptor blockade mechanism, the ubiquitin-mediated (Notch and Wnt) pathways, and the MAPK, PI3-K/Akt, NK-κB and STAT pathways, which can provide us with important insights into the roles of LMP2A in the EBV-associated latency state and various malignancies.     

Role of latent membrane protein 2 isoforms in Epstein-Barr virus latency

The oncogenic Epstein-Barr virus (EBV) infects the majority of the human population without doing harm and establishes a latent infection in the memory B-cell compartment. To accomplish this, EBV hijacks B-cell differentiation pathways and uses its own viral genes to interfere with B-cell signalling to achieve life-long persistence. EBV latent membrane protein 2A (LMP2A) provides a surrogate B-cell receptor signal essential for cell survival and is believed to have a crucial role in the maintenance of latency by blocking B-cell activation which would otherwise lead to lytic EBV infection. These two functions demand tight control of LMP2A activity and expression levels. Based on recent insights in the function of LMP2B, an isoform of LMP2A, we propose a model for how LMP2B modulates the activity of LMP2A contributing to maintenance of EBV latency.     

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-α) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.     

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1

Tumor necrosis factor (TNF) is a potent multi-functional cytokine with a homeostatic role in host defence. In case of deregulation, TNF is implicated in numerous pathologies. The latent membrane protein-1 (LMP1) is expressed by Epstein–Barr virus during viral latency and displaying properties of a constitutively activated member of the TNF receptor family. Both TNFR1 and LMP1 share a similar set of proximal adapters and signalling pathways although they display different biological responses. We previously demonstrated that the intracellular part of LMP1, LMP1-CT, a dominant-negative form of LMP1, inhibits LMP1 signalling.Here, we developed shorter versions derived from C-terminal part of LMP1 to investigate their roles on LMP1 and TNF signalling. We constructed several mutants of LMP1 containing a part of cytoplasmic signalling region fused to the green fluorescent protein. These mutants selectively impair signalling by LMP1 and TNF but not by IL-1β which uses other adapters. Dominant-negative effect was due to binding and sequestration of LMP1 adapters RIP, TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. Expression of these mutants impairs the recruitment of these adapters by TNFR1 and TNF-associated phenotypes. These mutants did not display cytostatic properties but were able to modulate TNF-induced phenotypes, apoptosis or cell survival, depending on the cell context. Interestingly, these mutants are able to inhibit a pro-inflammatory response in endothelial cells. These data demonstrate that LMP1 derived molecules can be used to design compounds with potential therapeutic roles in diseases due to TNF overactivation.     

Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma

Viral proteins expressed by EBV-associated tumors provide target Ags for immunotherapy. Adoptive T cell therapy has proven effective for posttransplant EBV-associated lymphoma in which all EBV latent Ags are expressed (type III latency). Application of immunotherapeutic strategies to tumors such as nasopharyngeal carcinoma and Hodgkin's lymphoma that have a restricted pattern of EBV Ag expression (type II latency) is under investigation. Potential EBV Ag targets for T cell therapy expressed by these tumors include latent membrane proteins (LMP) 1 and 2. A broad panel of epitopes must be identified from these target Ags to optimize vaccination strategies and facilitate monitoring of tumor-specific T cell populations after immunotherapeutic interventions. To date, LMP2 epitopes have been identified for only a limited number of HLA alleles. Using a peptide library spanning the entire LMP2 sequence, 25 CTL lines from patients with EBV-positive malignancies expressing type II latency were screened for the presence of LMP2-specific T cell populations. In 21 of 25 lines, T cell responses against one to five LMP2 epitopes were identified. These included responses to previously described epitopes as well as to newly identified HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-, B*3501-, B53-, and HLA-DR-restricted epitopes. Seven of the nine newly identified epitopes were antigenically conserved among virus isolates from nasopharyngeal carcinoma tumors. These new LMP2 epitopes broaden the diversity of HLA alleles with available epitopes, and, in particular, those epitopes conserved between EBV strains provide valuable tools for immunotherapy and immune monitoring.     

Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.