Virus Latent Membrane Protein 2A Research Articles

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) Enhances ATP Production in B Cell Tumors through mTOR and HIF-1α.

Epstein-Barr Virus (EBV) exists in a latent state in 90% of the world's population and is linked to numerous cancers, such as Burkitt's Lymphoma, Hodgkin's, and non-Hodgkin's Lymphoma. One EBV latency protein, latency membrane protein 2A (LMP2A), is expressed in multiple latency phenotypes. LMP2A signaling has been extensively studied and one target of LMP2A is the mammalian target of rapamycin (mTOR). Since mTOR has been linked to reprogramming tumor metabolism and increasing levels of hypoxia-inducible factor 1 α (HIF-1α), we hypothesized that LMP2A would increase HIF-1α levels to enhance ATP generation in B lymphoma cell lines. Our data indicate that LMP2A increases ATP generation in multiple Burkitt lymphoma cell lines that were dependent on HIF-1α. Subsequent studies indicate that the addition of the mTOR inhibitor, rapamycin, blocked the LMP2A-dependent increase in HIF-1α. Further studies demonstrate that LMP2A does not increase HIF-1α levels by increasing HIF-1α RNA or STAT3 activation. In contrast, LMP2A and mTOR-dependent increase in HIF-1α required mTOR-dependent phosphorylation of p70 S6 Kinase and 4E-BP1. These findings implicate the importance of LMP2A in promoting B cell lymphoma survival by increasing ATP generation and identifying potential pharmaceutical targets to treat EBV-associated tumors.

The impact of Epstein-Barr virus latent membrane protein 2A on the production of B cell activating factor of the tumor necrosis factor family (BAFF), APRIL and their receptors.

IntroductionEpstein‐Barr virus (EBV) establishes a lifelong infection in human B cells where the virus consistently expresses Latent Membrane Protein 2A (LMP2A) to promote B cell survival. A prior study indicates that LMP2A may increase the production of the pro‐survival factor, B cell Activating Factor of the tumor necrosis factor family (BAFF), which could also indirectly increase B cell survival. The current study sought to extend these findings and determine if LMP2A increased BAFF production and/or the responsiveness of LMP2A‐expressing cells to this cytokine.MethodsFour independently derived LMP2A‐negative and ‐positive B cell lymphoma cell lines were analyzed for BAFF and APRIL levels by both ELISA and Western Blot analysis. Additionally, flow cytometric analysis measured any LMP2A‐dependent changes in the receptors for BAFF and APRIL (BAFF‐R, transmembrane activator and calcium‐modulator and cyclophilin ligand interactor [TACI], B cell maturation antigen [BCMA]) in both LMP2A‐negative and ‐positive B cell lymphoma cell lines.ResultsIn contrast to previous reports, our data indicate that LMP2A does not increase the expression of BAFF or APRIL by Western blot analysis or ELISA. Additionally, flow cytometric analysis indicates that LMP2A does not influence the expression of the receptors for BAFF and APRIL: TACI, BAFF‐R, and BCMA.ConclusionTherefore, these data suggest that while EBV utilizes other latency proteins to regulate BAFF production, EBV does not appear to use LMP2A to enhance BAFF‐or APRIL‐dependent survival to promote EBV latency.

Two Pathways of p27Kip1 Degradation Are Required for Murine Lymphoma Driven by Myc and EBV Latent Membrane Protein 2A.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and MYC transgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressing MYC (λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1 is present at significantly lower levels in LMP2A/λ-MYC mice due to increased posttranslational degradation. P27Kip1 degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10) or in the nucleus via the SCFSkp2 complex, which depends on Cks1. We previously demonstrated an S10A knock-in of p27Kip1 (p27S10A/S10A) that prevented S10 phosphorylation failed to significantly delay tumor onset in LMP2A/λ-MYC mice. We also previously demonstrated that a Cks1 knockout partially delayed tumor onset in LMP2A/λ-MYC mice, but onset was still significantly faster than that in λ-MYC mice. Here, we have combined both genetic manipulations in what we call p27Super mice. LMP2A/λ-MYC/p27Super mice and λ-MYC/p27Super mice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Super mice was later than that in λ-MYC mice and not significantly different from that in λ-MYC/p27Super mice. The p27Super genotype also normalized G1-S-phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYC mice. Our results reveal both major pathways of p27Kip1 degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.IMPORTANCE BL is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain EBV, and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression of MYC Mice engineered to express EBV LMP2A along with MYC (LMP2A/λ-MYC mice) develop tumors much more quickly than mice only expressing MYC (λ-MYC mice). Blocking degradation of the cell cycle inhibitor protein p27Kip1 in LMP2A/λ-MYC mice causes tumors to develop later than in λ-MYC mice, showing that p27Kip1 degradation may play a larger role in EBV-positive BL than EBV-negative BL. Furthermore, our studies suggest the cell cycle is an attractive target as a treatment option for LMP2A-positive cancers in humans.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) increases IL-10 via Bruton’s tyrosine kinase and STAT3: Effect on T cell function

Abstract Data from our laboratory demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) promotes B cell lymphoma survival by enhancing IL-10 production, which has important implications in the treatment of EBV-associated B cell tumors. To determine the pathway used by LMP2A to increase IL-10, pharmacological inhibitors were added to LMP2A-expressing B cells and IL-10 was assessed. Our findings indicate for the first time, that LMP2A activates BTK to phosphorylate STAT3 in B cell tumors, which mediates the LMP2A-dependent increase in IL-10. Additionally, we assessed whether the LMP2A-mediated increase in IL-10 is immunosuppresssive to cytotoxic T cells that are critical for controlling the growth of EBV-associated tumors. Purified human CD8+ T cells activated with anti-CD3/CD28 mAbs were exposed to supernatants from either LMP2A-negative or –positive B cell lines for 48 hours. Our findings indicate that there is no difference in proliferation and IFN-γ secretion by activated CD8+ T cells exposed to supernatants from LMP2A-positive B cell lymphomas when compared to CD8+ T cells exposed to supernatants from LMP2A-negative B cell lymphomas. Although the LMP2A-mediated increase in IL-10 does not significantly influence the CD8+ T cell response, these findings highlight the possibility of using BTK and STAT3 inhibitors in clinical trials to treat EBV-associated lymphomas that express LMP2A. This work was supported by NIH grant 1R15CA149690-01.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 production through the activation of Bruton's tyrosine kinase and STAT3

Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and –positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A.

Epstein–Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation

Epstein–Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Epstein–Barr virus latent membrane protein 2A mediated activation of Sonic Hedgehog pathway induces HLA class Ia downregulation in gastric cancer cells

One of the immune evasion strategies manifested by malignant cells is the downregulation of the Human Leukocyte Antigen (HLA). HLA Class I (HLA- A, -B, -C) present endogenous peptides including viral and tumor antigens to cytotoxic T lymphocytes for immune mediated destruction. We have found the Epstein Barr Virus (EBV) Latent Membrane Protein 2A (LMP2A) to be responsible for this HLA downregulation in gastric cancer cells. Our results further indicate the Sonic Hedgehog pathway; primarily Gli1 to bring about the LMP2A mediated decrease in HLA expression.

Epstein-Barr virus latent membrane protein 2 induces autophagy to promote abnormal acinus formation.

Epstein-Barr virus latent membrane protein 2A (LMP2A) induces many characteristics of carcinoma, including transformation, migration, invasion, and impaired differentiation. The MCF10A cell line differentiates to form hollow acini when grown in Matrigel, and expression of LMP2A inhibited differentiation and anoikis induced by loss of matrix attachment. LMP2A-infected cells formed large, lobular structures rather than hollow acini. Autophagy inhibitors impaired this abnormal growth and induced caspase 3 activation and acinus formation. LMP2A also increased autophagosome formation and expression of proteins in the autophagosome pathway. These findings suggest that LMP2A may inhibit anoikis and luminal clearance in acini through induction of autophagy.

The B subunit of Escherichia coli enterotoxin helps control the in vivo growth of solid tumors expressing the Epstein-Barr virus latent membrane protein 2A.

Latent membrane protein 2A (LMP2A) is expressed on almost all Epstein–Barr virus (EBV)-associated tumors and is a potential target for immunotherapeutic intervention and vaccination. However, LMP2A is not efficiently processed and presented on major histocompatibility antigens class I molecules to generate potent cytotoxic T-lymphocytes (CTL) responses capable of killing these tumors. The B subunit of Escherichia coli enterotoxin (EtxB), causes rapid internalization and processing of membrane-bound LMP2A on EBV-infected B cells, and facilitates loading of processed-LMP2A peptides onto MHC class I. This re-directed trafficking/delivery of LMP2A to the MHC class I machinery enhances recognition and killing by LMP2A-specific CTL in vitro. To test the potential of EtxB to enhance immune targeting of LMP2A expressed in solid tumors, we generated a murine tumor model (Renca-LMP2A), in which LMP2A is expressed as a transgenic neoantigen on a renal carcinoma (Renca) cell line and forms solid tumors when injected subcutaneously into BALB/c mice. The data show that in BALB/c mice which have only low levels of peripheral Kd-LMP2A-specific CD8+ T cells, merely a transient inhibition of tumor growth is achieved compared with naïve mice; suggesting that there is suboptimal LMP2A-specifc CTL recognition and poorly targeted tumor killing. However, importantly, treatment of these mice with EtxB led to a significant delay in the onset of tumor growth and significantly lower tumor volumes compared with similar mice that did not receive EtxB. Moreover, this remarkable effect of EtxB was achieved despite progressive reduction in tumor expression of LMP2A and MHC class I molecules. These data clearly demonstrate the potential efficacy of EtxB as a novel therapeutic agent that could render EBV-associated tumors susceptible to immune control.

Epstein-Barr virus latent membrane protein 2A suppresses the expression of HER2 via a pathway involving TWIST and YB-1 in Epstein-Barr virus-associated gastric carcinomas.

To explore HER2 expression in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) and the possible mechanisms causing down-regulation of HER2 expression in EBVaGC, we first evaluated HER2 and LMP2A expression on a clinicopathological-features matched cohort including 78 EBVaGC and 216 EBV-negative gastric carcinoma (EBVnGC) cases by immunohistochemistry. Cases with high HER2 expression in EBVaGC were significantly less than in EBVnGC (5.1% versus 23.7%; p<0.001), and none of the 34 LMP2A+ EBVaGC showed high HER2 expression. Further, overexpressing LMP2A in EBV-negative SGC7901 cells significantly decreased HER2, TWIST and YB-1 mRNA by 36.1%±8.1%, 87.6%±14.0% and 83.8%±5.7%, and protein by 44%, 57% and 49%, respectively. Additionally, the nucleus/cytoplasm ratios of TWIST and YB-1 were also decreased by 85% and 80%, respectively. Silencing LMP2A by siRNA in EBV-positive SNU719 cells for 48 h significantly increased HER2, TWIST and YB-1 mRNA to 276.7%±14.6%, 1284.8%±38.2% and 332.0%±15.5% and protein to 212%, 457% and 232%, respectively. The nucleus/cytoplasm ratios of TWIST and YB-1 were up-regulated by 4.00- and 3.57-fold, respectively, following LMP2A down-regulation. Moreover, LMP2A+/HER2low EBVaGC cases presented the best overall survival compared with LMP2A-/HER2low and LMP2A-/HER2high cases (p=0.003, log-rank test). These results suggest that LMP2A may suppress the HER2 expression through the TWIST/YB-1 axis in EBVaGC.

Epstein–Barr virus latent membrane protein-2A alters mitochondrial dynamics promoting cellular migration mediated by Notch signaling pathway

Recently, migration and invasion of breast cancer cells have been linked with dysregulated mitochondrial dynamics. Mitochondria are essential cellular organelles that undergo continuous dynamic cycles of fission and fusion. It has been proposed that a delicate balance between these two processes is important for many pathophysiological outcomes including cancer. Epstein-Barr virus (EBV) is a gamma herpesvirus that is associated with various lymphoid and epithelial malignancies. The viral latent membrane protein 2A (LMP2A) has been shown to increase the invasive ability and induce epithelial-mesenchymal transition in nasopharyngeal carcinoma. Our present study reveals that mitochondrial dynamics also plays a critical role in Epstein-Barr virus-associated epithelial cancers. Our data indicate that viral LMP2A causes an elevated mitochondrial fission in gastric and breast cancer cells, which is manifested by elevated fission protein dynamin-related protein 1 (Drp1). Furthermore, LMP2A-mediated Notch pathway is responsible for this enhanced fission since inhibitors of the pathway decrease the expression of Drp1.

Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP₁₈₅₋₂₀₆. However, ex-vivo T cell proliferation to PLP₁₈₅₋₂₀₆ peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.

Epstein-Barr virus latent membrane protein 2A enhances MYC-driven cell cycle progression in a mouse model of B lymphoma

AbstractElevated expression of MYC is a shared property of many human cancers. Epstein-Barr virus (EBV) has been associated with lymphoid malignancies, yet collaborative roles between MYC and EBV in lymphomagenesis are unclear. EBV latent membrane protein 2A (LMP2A) functions as a B-cell receptor (BCR) mimic known to provide survival signals to infected B cells. Co-expression of human MYC and LMP2A in mice (LMP2A/λ-MYC) accelerates B lymphoma onset compared with mice expressing human MYC alone (λ-MYC mice). Here we show a novel role of LMP2A in potentiating MYC to promote G1-S transition and hyperproliferation by downregulating cyclin-dependent kinase inhibitor p27kip1 in a proteasome-dependent manner. Expressing a gain-of-function S10A mutant of p27kip1 has minor effect on tumor latency. However, pretumor B cells from λ-MYC mice expressing homozygous S10A mutant show a significant decrease in the percentage of S-phase cells. Interestingly, LMP2A is able to counteract the antiproliferative effect of the S10A mutant to promote S-phase entry. Finally, we show that LMP2A expression correlates with higher levels of MYC expression and suppression of p27kip1 before lymphoma onset. Our study demonstrates a novel function of EBV LMP2A in maximizing MYC expression, resulting in hyperproliferation and cellular transformation into cancer cells in vivo.

WW domain-containing oxidoreductase is involved in upregulation of matrix metalloproteinase 9 by Epstein–Barr virus latent membrane protein 2A

WW domain-containing oxidoreductase (WOX1) participates in tumor suppression and many other biologic functions, but its molecular and functional interactions with viral proteins remain largely unknown. This study reveals that WOX1 is physically associated with latent membrane protein 2A (LMP2A), an oncoprotein of Epstein–Barr virus. The molecular interaction involves the tyrosine residue 33 of WOX1 and the proline-rich motifs of LMP2A. Interestingly, endogenous WOX1 is required for some LMP2A-triggered, cancer-promoting effects, including activation of extracellular signal-regulated kinase-1/2, upregulation of matrix metalloproteinase 9 (MMP9) and promotion of cell invasion. Upon knockdown of endogenous WOX1, LMP2A-triggered MMP9 induction is restored by exogenous wild-type WOX1, but not by a WOX1 mutant defective in LMP2A binding. These results indicate that, through interaction with LMP2A, WOX1 is involved in MMP9 induction, suggesting a novel role of WOX1 in Epstein–Barr virus-associated cancer progression.

Enrichment of stem-like cell population comprises transformation ability of Epstein–Barr virus latent membrane protein 2A for non-transformed cells

Epstein–Barr virus (EBV) is a representative human oncogenic virus that causes malignancies of various cell lineages. LMP2A, an EBV-encoded latent membrane protein, is expressed in EBV-associated malignancies of various cell lineages. LMP2A caused visible tumor formation transplanted in nude mice when transferred to immortalized non-transformed fibroblasts, NIH3T3. LMP2A-expressing cells showed higher ability of colony formation in soft agar than empty vector-transfected control cells, although the expression of LMP2A did not cause focus transformation in low serum concentrations. LMP2A expression increased the size of Hoechst 33,342 dye excreting side population (SP), in which cancer-initiating cells or cancer stem-like cells were enriched. SP increase by LMP2A was also responsible for colony formation in soft agar. The LMP2A-mediated SP increase depended on the activations of Stat3, MEK/ERK, and PI3K pathways, and on upregulation of HMGA2. Enrichment of SP, stem-like cells, by LMP2A promoted the transformation capability of LMP2A from non-transformed cells. The enrichment of stem-like cell population by a virus-encoded factor might explain the oncogenic functions of oncogenic viruses.

Effects of Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) on cytokine production in primary and transformed B cells (105.40)

Abstract Epstein-Barr Virus (EBV) latently-infected B cells are precursors of EBV-associated malignancies. However, the transition from a latently-infected cell to a malignant tumor cell is poorly understood. Studies show that EBV modulates pro-survival and anti-inflammatory cytokine levels in EBV-transformed cell lines. However, gene expression in these cell lines may not reflect gene expression found in latent B cells and lymphomas. One EBV protein, LMP2A, is consistently detected in both latently-infected resting B cells, as well as in EBV-associated malignancies. Therefore, we hypothesized that LMP2A may modulate cytokines that aid in the transition between latency and malignancy. Cytokine array data analyzing B cells from LMP2A-transgenic mice and B cell lymphomas expressing LMP2A indicate that LMP2A does not globally alter B cell-produced cytokine levels, but rather targets a limited number of cytokines. Additional studies using ELISA and Real-time RT-PCR confirm that LMP2A increases IL-10 levels and flow cytometry reveals that LMP2A increases the amount of IL-10 produced per B cell. Inhibitors of the LMP2A signaling pathway are being tested to determine the signaling requirements for LMP2A-mediated increases in IL-10. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumor survival and anti-tumor responses.

Epstein-Barr virus latent membrane protein 2A promotes invasion of nasopharyngeal carcinoma cells through ERK/Fra-1-mediated induction of matrix metalloproteinase 9.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is highly metastatic, and this malignant feature may be promoted by an EBV oncoprotein, latent membrane protein 2A (LMP2A). Acting as a signal regulator, LMP2A can enhance invasiveness and motility of epithelial cells. Downstream from the LMP2A-triggered signaling events, it is largely unknown what key effector proteins are induced and essentially promote cell invasion. In the present study, we found that in NPC cells, LMP2A upregulated matrix metalloproteinase 9 (MMP9), a metastasis-associated protease. LMP2A increased MMP9 expression at both the mRNA and protein levels. It also activated the MMP9 promoter, in which two AP-1 elements were required for the promoter activation. Among AP-1 transcription factors, Fra-1 was induced by LMP2A and is essential for LMP2A-triggered MMP9 expression. Induction of Fra-1 was dependent on the LMP2A-activated ERK1/2 pathway, and induction of the ERK1/2-Fra-1-MMP9 axis required PY motifs in the amino-terminal domain of LMP2A. Notably, LMP2A-promoted invasion of NPC cells was blocked when MMP9 expression, Fra-1 induction, or ERK1/2 activation was inhibited. In addition, we found an association of LMP2A with MMP9 expression in NPC tumor biopsy specimens, where Fra-1 was a major mediation factor. This study reveals an underlying mechanism of LMP2A-induced cell invasion, from signal transduction to upregulation of a critical protease. Considering that MMP9 can also be upregulated by another EBV oncoprotein, LMP1, this protease may be a pivotal effector at which the EBV-induced, invasion-promoting mechanisms converge, serving as an attractive therapeutic target for NPC treatment.

Reconstituted complexes of mycobacterial HSP70 and EBV LMP2A-derived peptides elicit peptide-specific cytotoxic T lymphocyte responses and anti-tumor immunity

Epstein–Barr virus (EBV) latent membrane protein 2A (LMP2A) is a subdominant antigen expressed in EBV-associated malignancies, such as Hodgkin's diseases (HD) and nasopharyngeal carcinoma. A large number of previous studies have described LMP2A as an ideal target antigen in immunotherapy of EBV-related diseases, while limited successes have been achieved in clinical trials. Mycobacterium tuberculosis heat shock protein 70 (MtHsp70) is known as an effective molecular adjuvant for protein- or epitope-based vaccines. In the present study, we reconstituted two chaperone complexes of MtHsp70 and LMP2A-derived peptides (LMP2A356–364 FLYALALLL and LMP2A426–434 CLGGLLTMV) in vitro. We then investigated LMP2A-specific immune responses induced by reconstituted complexes of MtHsp70 and LMP2A-peptides using both EBV infected healthy donor PBMCs and HLA-A2.1 transgenic mouse models. We found that reconstituted complexes of MtHsp70 and LMP2A-peptides significantly elicit LMP2A-specific IFN-γ-producing cells and rousted cytotoxic T lymphocytes (CTLs) in vitro and in vivo. In addition, LMP2A-specific immune responses induced by the reconstituted complexes of MtHsp70 and LMP2A-peptides mediated potently protective activity as well as therapeutic efficacy against LMP2A-expressed tumor challenge in mouse models. These studies provide new insights for the development of novel LMP2A-based vaccines against EBV-associated malignancies.

Widespread sequence variation in the Epstein-Barr virus latent membrane protein 2A gene among northern Chinese isolates

Latent membrane protein 2A (LMP2A) is expressed in most Epstein-Barr virus (EBV)-associated malignancies. Besides its roles in the maintenance of latent infection and epithelial-cell transformation, LMP2A could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence polymorphisms in LMP2A from northern Chinese EBV-associated gastric carcinoma patients, nasopharyngeal carcinoma patients and healthy donors were identified and compared with the prototype B95-8 strain. Four consistent mutations were detected in all isolates. Frequent mutations in the analysed sequences distinguished two and seven types of sequence variation in exon 1 and exons 2-8, respectively, with no consistent association shown between the genotyping of the two gene fragments. The immunoreceptor tyrosine-based activation motif and PY motif in the amino terminus were strictly conserved. Nine of the 16 identified CTL epitopes were affected by at least one point mutation, which may confer complexity to proposed immunotherapeutic approaches for EBV-associated malignancies. Most changed epitopes showed higher mutation rates in tumour isolates than in throat-washing samples from healthy donors, in accordance with the idea that virus strains can evade immune surveillance by altering amino acids within LMP epitopes. This first detailed investigation of sequence variations in the LMP2A gene reveals classifiable sequence polymorphisms in exon 1 and exons 2-8, and encourages further work on the impact of viral gene variations on tumour persistence and CTL-based immunotherapy.

Epstein-Barr Virus Latent Membrane Protein 2A protects B cells from MYC-induced apoptosis and accelerates tumor development (45.14)

Abstract Despite its initial isolation from Burkitt's lymphoma (BL) tumors over 40 years ago, the exact contribution of Epstein-Barr Virus (EBV) to BL is undefined. EBV encodes for multiple proteins in latently-infected B cells that affect B cell survival and activation. Due to the ability of one of these latency proteins, latent membrane protein 2A (LMP2A) to protect B cells from apoptosis, we tested if LMP2A protects B cells from apoptosis induced by aberrant c-MYC expression that precedes and dominates BL. We crossed LMP2A-transgenic mice (LMP2A-Tg) in which all B cells express LMP2A to a transgenic mouse that expresses a BL translocation of myc (λ-MYC-Tg mice). LMP2A protects B cells from MYC-induced apoptosis and accelerates the development of B cell lymphomas in LMP2A/λ-MYC-Tg mice. LMP2A also increases the expression of Bcl-XL in both pre-tumor B cells and tumor cells, suggesting a mechanism for LMP2A mediated B cell survival in the presence of MYC. These results support a hypothesis that EBV LMP2A promotes tumor development by protecting pre-tumor B cells that would normally apoptose after the c-myc translocation. This work is supported by Public Health Service grants CA62234 and CA73507 from the NCI.