EBV-encoded Latent Membrane Protein Research Articles

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) Induces the Expression of the Cellular MicroRNA miR-146a

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g. the “vault” RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is up-regulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NF-κB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NF-κB-response elements. Conversely, a LMP1-mutant deficient in NF-κB-activation failed to activate the promoter. The “CAO”-LMP1 variant which has an increased potential to induce NF-κB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt’s lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt’s lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells up-regulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

Epstein-Barr virus-encoded LMP1 promotes cisplatin-induced caspase activation through JNK and NF-κB signaling pathways

Our previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) potentiates chemotherapeutic agent-induced apoptosis in human cell lines of epithelial origin: cervical carcinoma-derived HeLa cells and nasopharyngeal carcinoma-derived TW03 cells. LMP1 acted upstream of caspase-dependent mitochondrial perturbation, and the effect was mapped to the C-terminal signaling domain of LMP1, designated CTAR2. CTAR2 is known to engage the c-Jun N-terminal kinase (JNK) and NF-κB pathways, and we show here that SP600125, a selective JNK inhibitor, suppresses LMP1 potentiation of cisplatin-induced mitochondrial damage and caspase activation in HeLa cells. Moreover, the potentiation of cisplatin-triggered caspase activation was blocked by Bay11-7082, a potent inhibitor of NF-κB. Similar results were obtained when a dominant negative form of IκB, a specific repressor of NF-κB, was co-expressed with LMP1. The current data support the notion that LMP1 modifies stress-induced apoptosis in epithelial cells through molecular interactions downstream of its C-terminal signaling domain.

Epstein–Barr virus latent membrane protein 2A and autoimmunity

Epstein-Barr virus (EBV) has been associated with autoimmune diseases for over 40 years. However, the mechanisms by which EBV might promote autoimmune development remain elusive. Many of the hypotheses for the means by which EBV might achieve this incorporate the idea that autoimmune responses are initially immune responses against EBV proteins that crossreact with endogenous human proteins. However, recent evidence using transgenic mouse models suggests that B cells expressing the EBV-encoded protein latent membrane protein 2A (LMP2A) bypasses normal tolerance checkpoints and enhances the development of autoimmune diseases. Evidence from transgenic mouse models supports a paradigm in which LMP2A could promote autoimmune development. This novel model provides a framework to test potential mechanisms by which EBV could promote the development of autoimmune responses and might enable the identification of strategies to treat EBV-associated autoimmune diseases.

The Up-regulation of Vimentin and Ezrin in the Epstein-Barr Virus LMP1 Gene Transfected Cells

Epstein-Barr virus (EBV) is associated with the development of a variety of highly metastatic carcinomas, including nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1) is essential for B-cell transformation. In this study, we used two-dimensional differential gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to study the mechanism behind tumor invasion and metastasis. Eight proteins, including Vimentin and Ezrin, were identified from the alteration of expressed proteins in HEK-293 cells responding to LMP1 gene transfection. Vimentin is a major protein of the mesenchymal intermediate filament, which maintains the cytoskeleton conformation. Ezrin is also an essential protein that links the cell membrane to the actin cytoskeleton. The up-regulation of Vimentin and Ezrin in the LMP1 gene-transfected cells suggests that EBV LMP1 is involved in the progression and metastasis of NPC.

Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral Latent Membrane Protein 1 and the immunomodulatory protein galectin 9

BackgroundNasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested.MethodsMalignant epithelial cells derived from NPC xenografts – LMP1-positive (C15) or negative (C17) – were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking.ResultsHLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1.ConclusionThis study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells.

Absence of Epstein-Barr virus infection in squamous cell carcinoma of upper urinary tract and urinary bladder

Objectives To address whether Epstein-Barr virus (EBV) infection may be involved in the carcinogenesis of squamous cell carcinoma of the upper urinary tract and urinary bladder (SCC-UB). EBV has been implicated in the genesis of a variety of human cancers, including urothelial carcinoma of the urinary bladder. Methods Whether EBV infection is related to SCC-UB carcinogenesis was investigated by in situ hybridization for EBV-encoded RNA and immunohistochemistry for latent membrane protein-1 in 26 cases of SCC-UB. Results EBV-encoded RNA and latent membrane protein-1 were identified in the control case of nasopharyngeal carcinoma. None of the SCC-UB cases had a nuclear signal of EBV-encoded RNA, and the cancer cells, normal urothelial cells, and inflammatory cells were all negative for latent membrane protein-1, irrespective of the site of SCC-UB. Conclusions This study is the first to explore the role of EBV infection in SCC-UB. Our results suggest that EBV infection is not involved in the carcinogenesis of SCC of the renal pelvis, ureter, and urinary bladder.

Upregulated expression of kappa light chain by Epstein–Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-κB and AP-1 pathways

B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1-targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-κB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-κB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins.

Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus encoded LMP-1

Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma characterized by consistent association with Epstein-Barr virus (EBV). Of the EBV-encoded product, latent membrane protein-1 (LMP-1) is considered to be an onco-protein playing an essential role in cell transformation and metastasis. In this study, we used a recombinant adeno-associated virus type 2 vector (rAAV-2) to deliver small hairpin RNA (shRNA) targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells and evaluated the effect of long-term suppression of LMP-1 on NPC growth and metastasis in vivo and in vitro. An NPC metastasis nude mouse model with NPC xenograft transplanted in liver was established. The NPC C666-1 cells infected with rAAV-shRNA-LMP-1 or rAAV-EGFP were inoculated in the livers of nude mice. Formation of liver and lung metastasis was evaluated at day 14 after tumor inoculation. Our results demonstrate that rAAV-shRNA-LMP-1 effectively infected C666-1 cells and suppressed LMP-1 expression. Such suppression, in turn, did not significantly inhibit tumor growth, but prevented NPC metastasis in the liver as well as in the lung. Consistent with in vivo data, the in vitro studies in NPC C666-1 cell cultures showed that suppression of LMP-1 by rAAV-shRNA-LMP-1 significantly reduced cell mobility and transmembrane invasion ability. These results demonstrated for the first time that long-term suppression of EBV-encoded LMP-1 in vivo is an effective means for preventing NPC metastasis.

EBV Latent Membrane Protein 2A Induces Autoreactive B Cell Activation and TLR Hypersensitivity

EBV is associated with systemic lupus erythematosus (SLE), but how it might contribute to the etiology is not clear. Since EBV-encoded latent membrane protein 2A (LMP2A) interferes with normal B cell differentiation and function, we sought to determine its effect on B cell tolerance. Mice transgenic for both LMP2A and the Ig transgene 2-12H specific for the ribonucleoprotein Smith (Sm), a target of the immune system in SLE, develop a spontaneous anti-Sm response. LMP2A allows anti-Sm B cells to overcome the regulatory checkpoint at the early preplasma cell stage by a self-Ag-dependent mechanism. LMP2A induces a heightened sensitivity to TLR ligand stimulation, resulting in increased proliferation or Ab-secreting cell differentiation or both. Thus, we propose a model whereby LMP2A induces hypersensitivity to TLR stimulation, leading to activation of anti-Sm B cells through the BCR/TLR pathway. These data further implicate TLRs in the etiology of SLE and suggest a mechanistic link between EBV infection and SLE.

Expression of syndecan-1 (CD138) in nasopharyngeal carcinoma is correlated with advanced stage and poor prognosis

Nasopharyngeal carcinoma (NPC) is an important Epstein-Barr virus-associated head and neck malignancy in Taiwan. Syndecan-1 (CD138) is involved in growth, differentiation, invasiveness, and metastatic potential of certain tumors, but its expression in NPC has never been studied. In this study, detection of expression of syndecan-1 protein and Epstein-Barr virus-encoded latent membrane protein-1 (LMP-1) in primary, recurrent, and metastatic NPC specimens in paraffin sections was performed by immunohistochemistry. The quantity of syndecan-1 messenger RNA in tumor cells was investigated by real-time reverse transcriptase polymerase chain reaction using laser capture microdissection. The results of immunohistochemical staining of syndecan-1 and LMP-1 correlated with clinicopathologic features of NPC. Eighteen (20.9%) of 86 primary, 9 (24.3%) of 37 recurrent, and 15 (44.1%) of 34 metastatic NPC samples were positive for syndecan-1, and 37 (43.0%) primary, 18 (48.6%) recurrent, and 12 (35.3%) metastatic samples were positive for LMP-1 expression. Primary NPCs with syndecan-1 protein expression were more frequently associated with advanced clinical stages and worse 5-year survival rates than those without (P = .015 and P = .0021, respectively). Conversely, the LMP-1 expression did not correlate with tumor stage or prognosis but occurred more often in nonkeratinizing carcinoma than keratinizing squamous cell carcinoma (unpublished observation). The inverse expression of syndecan-1 and LMP-1 was noted in primary NPC specimens (total 4/18 versus 35/68, P = .05). The reverse transcriptase polymerase chain reaction revealed low syndecan-1 messenger RNA levels in both primary and metastatic NPC. In conclusion, the protein expression of syndecan-1 in 21% of primary NPC was associated with advanced disease and poor prognosis, and the protein expression correlated with transcription levels.

IL-10 can induce the expression of EBV-encoded latent membrane protein-1 (LMP-1) in the absence of EBNA-2 in B lymphocytes and in Burkitt lymphoma- and NK lymphoma-derived cell lines

AbstractEBV-positive nasopharyngeal carcinoma and Hodgkin, T, and natural killer (NK) lymphomas express EBNA-1 and the latent membrane proteins (LMP1-2; type II latency). In contrast to type III EBV-transformed lymphoblastoid cell lines, in these cells the LMPs are expressed in the absence of EBNA-2. We have previously reported that exposure to CD40 ligand and IL-4 could induce LMP-1 in an in vitro EBV-infected Hodgkin lymphoma-derived cell line, which expressed only EBNA-1. We show now that both human and EBV-encoded IL-10 can induce LMP-1 in the absence of EBNA-2 in the Daudi, P3HR1, and other BL cell lines. Interestingly, induction of LMP-1 was not accompanied by the downregulation of BCL-6. IL-10 could also induce LMP-1 in the conditional lymphoblastoid cell line ER/EB2-5 where EBNA-2 was downregulated in the absence of estrogen. Moreover, IL-10 could induce the expression of LMP-1 in tonsillar B cells infected with the nontransforming, EBNA-2-deficient EBV strain P3HR1 and enhance LMP-1 expression in 2 EBV-positive NK lymphoma lines. The demonstration that IL-10 can induce the expression of LMP-1 in an EBNA-2-independent manner shows that the major transforming EBV gene LMP-1 can be induced by extracellular signals in lymphoid cells, and IL-10 might contribute to the establishment of type II EBV latency.

Downregulation of Epstein-barr Virus-Encoded Latent Membrane Protein-1 by Arsenic Trioxide in Nasopharyngeal Carcinoma Cells

It was documented that nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and that EBV-encoded latent membrane protein-1 expression (LMP1) plays an important role in the pathogenesis of NPC. In preclinical studies, arsenic trioxide (As2O3) has been identified as a promising anticancer agent for treatment of NPC. The purpose of this study is to investigate if this agent can inhibit the expression of LMP1 and therefore lead to growth inhibition of NPC cells in vitro. LMP1-positive NPC cells, HNE1-LMP1, were treated with 3 micromol/L of As2O3 for 96 hours. The LMP1 protein expression and mRNA level in HNE1-LMP1 cells were determined by western blot, confocal immunofluorescence staining and semiquantitative reverse transcriptase reaction (RT-PCR). Apoptosis was determined by light microscopy and the TUNEL method. Alterations in the cell cycle distribution were also investigated by flow cytometry. MTT assay and colony formation assay were used to detect the proliferation of the cells. The LMP1-negative parental cell lines HNE1 and HNE2 were used as control in an attempt to elucidate the role of LMP1 in the anticancer effect of As2O3 on NPC cells. The expression of LMP1 at the protein and mRNA level was reduced after exposure to 3 micromol/L As2O3. This dose of As2O3 significantly induced apoptosis and growth retardation of HNE1-LMP1 cells. In addition, more HNE1-LMP1 cells were induced to G0/G1 and G2/M arrest. The same dose of As2O3 had a moderate effect on HNE1 and HNE2 cells. Arsenic trioxide can inhibit LMP1 expression and dictate apoptosis and alterations of cell cycle distribution as well as growth retardation. LMP1-positive NPC cells are more sensitive to As2O3 treatment than LMP1-negative NPC cells.

Identification of novel phosphoproteins in signaling pathways triggered by latent membrane protein 1 using functional proteomics technology

Previous studies have shown that the Epstein-Barr virus-encoded latent membrane protein1 (LMP1) could activate nuclear factor kappa B, activator protein-1, and Janus kinases/signal transducer and activation of transcription factors pathways. However, many signaling molecules and downstream target proteins triggered by LMP1 have not been identified. To determine the functional components in signaling pathways triggered by LMP1, we combined the novel strategy of phosphoprotein enrichment with proteomics technology to elucidate the signaling cascade activated by LMP1. We found that LMP1 could increase the quantity of total phosphoproteins by 18.03%, and 43 proteins showed significant changes in the degree of phosphorylation when LMP1 was expressed. Twenty-five signaling molecules or downstream targets of signaling pathways triggered by LMP1 were identified, several of which had previously been implicated in LMP1 signal pathways. The other proteins, including annexin A2, heat shock protein 27, stathmin, annexin I, basic transcription factor 3, and porin, were novel signaling molecules or targets with no previously known function in LMP1 signal transduction. The method used here has proven to be suitable for the identification of molecules involved in various signaling pathways.

The C-terminal Activating Region 2 of the Epstein-Barr Virus-encoded Latent Membrane Protein 1 Activates NF-κB through TRAF6 and TAK1

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-kappaB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-kappaB activation, with CTAR2 being the main NF-kappaB activator. Although the CTAR1-mediated NF-kappaB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-kappaB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-kappaB pathways, the CTAR2-mediated NF-kappaB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-kappaB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-kappaB: a major one through CTAR2/TRAF6/TAK1/IKKbeta (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKalpha (noncanonical pathway).

Bortezomib (Velcade™) and the Bcl-2 Inhibitor HA14-1 Synergistically Stimulate Apoptosis in EBV-Transformed Lymphocytes: A Potential Approach to Treatment of EBV-Associated Lymphoproliferative Disorders.

Epstein-Barr virus (EBV) transforms B-cells into immortalized lymphoblastoid cell lines (LCLs) by triggering signaling pathways that lead to the activation of multiple transcription factors, including NF-kappaB. EBV appropriation of the NF-kappaB pathway via EBV-encoded latent membrane protein-1 plays a pivotal role in EBV-mediated up-regulation of cellular growth-promoting and anti-apoptotic genes, including Bcl-2. Proteosome inhibitors provide a possible approach to inhibit constitutive activation of NF-kappaB in EBV-transformed B-cells. To determine whether the proteosome inhibitor bortezomib (PS-341, Velcade™) may have potential as a therapeutic anti-tumor agent in EBV-driven B-cell neoplasms, we evaluated the effect of bortezomib in EBV-immortalized LCLs (Sweig) in the presence and absence of the small molecular inhibitor of Bcl-2, HA14-1. Sweig cells were treated with increasing concentrations of bortezomib (1–1000 nM) for 24 hrs in the absence and presence of HA14-1 (10 uM), and assayed for cell proliferation (by MTT assay), apoptosis (by quantitation of DNA fragmentation in a flow cytometric TUNEL assay), caspase activation (by Western blotting), and levels of Bcl-2 family proteins (by Western blotting). Bortezomib alone inhibited proliferation of LCLs in a dose-dependent manner with an IC50 of 50 nM. At this concentration, bortezomib stimulated apoptosis in only 20% of LCLs, and we observed minimal caspase-3 cleavage. HA14-1 treatment alone had no significant effects on proliferation, apoptosis, or caspase-3 cleavage. When the Bcl-2 inhibitor HA14-1 was added to bortezomib, we observed marked enhancement of anti-proliferative and pro-apoptotic effects in LCLs. This synergistic interaction was observed with sequential exposure of LCLs to bortezomib followed by HA14-1 (8 hrs) or simultaneous exposure to both drugs for 24 hrs, but not when HA14-1 was added 8 hrs prior to bortezomib. In the presence of HA14-1, the IC50 for bortezomib decreased to 10 nM. Bortezomib (25 nM)/HA14-1 stimulated apoptosis in 80% of LCLs compared to bortezomib alone (20%). Moreover, bortezomib/HA14-1 triggered pronounced cleavage of both caspase 9 and 8, as well as caspase 3, suggesting activation of apoptosis through both mitochondrial and extrinsic pathways. Interestingly, Bcl-2 protein levels were increased, with appearance of the pro-apoptotic Bcl-2 cleavage fragment, in LCLs treated with bortezomib/HA14-1. These studies have demonstrated for the first time that bortezomib mediates anti-proliferative and pro-apoptotic effects in EBV-transformed lymphocytes, and that these effects are dramatically enhanced in the presence of a Bcl-2 inhibitor. These findings support further investigation of bortezomib in EBV-associated lymphoproliferative diseases and suggest that combining bortezomib with Bcl-2 antagonists, such as rituximab or Bcl-2 antisense oligonucleotides, may enhance anti-tumor efficacy. Studies are in progress to further delineate the mechanism(s) of interaction between bortezomib and HA14-1 in LCLs, and to evaluate the anti-tumor efficacy of bortezomib in the pre-clinical SCID/human model of EBV-associated lymphoproliferative disease.

Constitutive activation of the CD40 pathway promotes cell transformation and neoplastic growth

CD40, a tumour necrosis factor (TNF) receptor family member, is expressed in a variety of cell types, including B lymphocytes, macrophages, fibroblasts, endothelial and epithelial cells, and this widespread expression is likely to account for its central role in normal physiology and disease pathogenesis. In this study, we provide evidence to support a role for constitutive CD40 signalling in cell transformation. We show that the ligand for CD40 (CD40L/CD154) is expressed in CD40-positive human breast tumour biopsies, suggesting that the constitutive activation of the CD40 receptor in vivo may contribute to the oncogenic process. Coexpression of CD40 and CD40L confers oncogenic effects on immortalized human epithelial cells in vitro, increasing their proliferation, motility and invasion. Expression of LMP:CD40, a hybrid molecule comprising the N-terminus and transmembrane domains of the Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) fused to the cytoplasmic tail of CD40, mimics a constitutively active CD40 receptor and promotes the transformation of immortalized rodent fibroblasts in vitro and their oncogenicity in vivo. The observed effects of aberrant CD40 activation on cell transformation are largely diminished upon suppression of the oncogenic NF-kappaB signalling pathway. Taken together, our results suggest a role for the constitutive engagement of the CD40L/CD40/NF-kappaB activation pathway in cell transformation and neoplastic growth. Strategies that neutralize this pathway may therefore be useful in cancer treatment and prevention.

Epstein–Barr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a variety of malignancies including nasopharyngeal carcinoma. The EBV-encoded latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced B-lymphocyte transformation and has been shown to transform rodent fibroblasts. LMP1 contains two signaling domains, the carboxy-terminal activating region 1 and 2 (CTAR1 and CTAR2), by which NF-kappaB, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase, and c-Jun N-terminal kinase are activated. In this study, the role of CTAR1 and CTAR2 in LMP1-mediated transformation of rodent fibroblasts was analysed. CTAR1 was found to be necessary for rodent fibroblast transformation, whereas CTAR2 was dispensable. The activation of the PI3K pathway in Rat-1 cells by LMP1 and LMP1-CTAR1 in transformed cells resulted in phosphorylated Akt and phosphorylated glycogen synthase kinase 3beta. The role of PI3K and NF-kappaB activation in LMP1-mediated transformation was further analysed using the chemical inhibitors LY294002 and BAY 11-7085. LY294002 inhibited CTAR1-induced focus formation and anchorage-independent growth, whereas BAY 11-7085 did not inhibit focus formation or anchorage-independent growth. Similar studies in human fibroblasts confirmed that LMP1-CTAR1 also mediates aberrant growth, phosphorylation of Akt, and decreased levels of p27. These findings indicate that LMP1-mediated rodent fibroblast transformation is dependent upon activation of PI3K and Akt and is independent of activation of NF-kappaB.

Roles of TNF Receptor-Associated Factor 3 in Signaling to B Lymphocytes by Carboxyl-Terminal Activating Regions 1 and 2 of the EBV-Encoded Oncoprotein Latent Membrane Protein 1

TNFR-associated factor (TRAF)3, an adaptor protein that binds the cytoplasmic domains of both CD40 and the EBV-encoded oncoprotein latent membrane protein (LMP)1, is required for positive signaling by LMP1 but not CD40 in B lymphocytes. The present study further investigated how TRAF3 participates in LMP1 signaling. We found that TRAF3 mediates signaling both through direct interactions with the C-terminal activating region (CTAR)1 of LMP1 and through indirect interactions with the CTAR2 region of LMP1 in mouse B cells. Notably, our results demonstrated that the CTAR2 region appears to inhibit the recruitment of TRAF1 and TRAF2 to membrane rafts by the CTAR1 region. Additionally, the absence of TRAF2 in B cells resulted in only a modest reduction in CTAR1-mediated signals and no detectable effect on CTAR2-mediated signals. CTAR1 and CTAR2 cooperated to achieve the robust signaling activity of LMP1 when recruited to the same membrane microdomains in B cells. Interestingly, TRAF3 deficiency completely abrogated the cooperation between CTAR1 and CTAR2, supporting the hypothesis that TRAF3 participates in the physical interaction between CTAR1 and CTAR2 of LMP1. Together, our findings highlight the central importance of TRAF3 in LMP1-mediated signaling, which is critical for EBV persistent infection and EBV-associated pathogenesis.

Secondary extension of Hodgkin's disease to the skin with positive staining for EBV-encoded latent membrane protein

Secondary extension of Hodgkin's disease to the skin with positive staining for EBV-encoded latent membrane protein

Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in EBV-associated nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) is associated with virtually all cases of undifferentiated nasopharyngeal carcinoma (NPC) and has been classified as a group I carcinogen. In addition to its potential role in the pathogenesis of NPC, EBV also provides a possible target for immunotherapy of NPC, since a limited number of viral genes are expressed in the neoplastic cells. The EBV-encoded latent membrane protein 2A (LMP2A) is considered a promising target since it provides epitopes recognized by EBV-specific T-cells. Using immunohistochemistry, the present study shows that LMP2A is expressed at the protein level in the neoplastic cells of 16 of 35 (45.7%) NPC biopsies. This finding provides further evidence suggesting that NPC tumour cells may be susceptible to lysis by cytotoxic T-cells directed against LMP2A and should encourage efforts to develop immunotherapeutic approaches for the treatment of NPC.