Latent Membrane Protein 1 Activation Research Articles

Assembly and activation of EBV latent membrane protein 1

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

Oligomerization analysis as a tool to elucidate the mechanism of EBV latent membrane protein 1 inhibition by pentamidine

Latent membrane protein 1 (LMP1) is a gene product of the Epstein-Barr virus (EBV), a widely spread virus present in 90–95% of the world's population. EBV can lead to several malignancies, in which LMP1 was shown to play a key role. LMP1 is active only in the oligomeric form and its fifth transmembrane domain (TMD-5) is critical for the oligomerization, with D150 identified as a key residue for LMP1 activation. Here we propose an NMR-based approach to treat the complex oligomerization equilibria with slow conformational exchange. Using this method we investigate the TMD-5 in DPC micelles. We show that the pKa of D150 equals 7.4. Uncharged form of TMD-5 associates into dimers and trimers, deprotonation of D150 induces the high-order oligomerization of the protein and enhances dramatically its trimerization. Pentamidine interacts mainly with the charged TMD-5, destroying the oligomers and stabilizing the monomer and trimer. Using computer simulations we investigate the structural basis of TMD-5/pentamidine interaction. Our data suggest that D150 is likely charged in the full-length LMP1 under native conditions.

Activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated lipogenesis by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) promotes cell proliferation and progression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element‐binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV‐infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1‐mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1‐mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo. Our findings demonstrate that LMP1 activation of SREBP1‐mediated lipogenesis promotes tumor cell growth and is involved in EBV‐driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required.

Interferon (IFN) regulatory factors (IRFs) have crucial roles in immune regulation and oncogenesis. We have recently shown that IRF4 is activated through c-Src-mediated tyrosine phosphorylation in virus-transformed cells. However, the intracellular signaling pathway triggering Src activation of IRF4 remains unknown. In this study, we provide evidence that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) promotes IRF4 phosphorylation and markedly stimulates IRF4 transcriptional activity, and that Src mediates LMP1 activation of IRF4. As to more precise mechanism, we show that LMP1 physically interacts with c-Src, and the phosphatidylinositol 3 kinase (PI3K) subunit P85 mediates their interaction. Depletion of P85 by P85-specific short hairpin RNAs disrupts their interaction and diminishes IRF4 phosphorylation in EBV-transformed cells. Furthermore, we show that Src is upstream of PI3K for activation of both IRF4 and Akt. In turn, inhibition of PI3K kinase activity by the PI3K-speicfic inhibitor LY294002 impairs Src activity. Our results show that LMP1 signaling is responsible for IRF4 activation, and further characterize the IRF4 regulatory network that is a promising therapeutic target for specific hematological malignancies.

High molecular weight complex analysis of Epstein–Barr virus Latent Membrane Protein 1 (LMP-1): Structural insights into LMP-1's homo-oligomerization and lipid raft association

LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein–Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1's TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers.

Epstein-Barr virus latent membrane protein 1 induces CD69 expression through activation of nuclear factor-κB

Latent membrane protein‑1 (LMP-1) of Epstein‑Barr virus (EBV) promotes tumorigenesis. Here, we report that LMP-1 activates the immunoregulatory molecule CD69 gene transcription through a nuclear factor-κB (NF-κB)‑dependent pathway. CD69 expression was upregulated in LMP-1‑expressing EBV-immortalized human B-cell lines and an EBV-positive Burkitt's lymphoma cell line. LMP-1 expression increased CD69 expression at the transcriptional level. CD69 promoter was regulated by LMP-1 activation of NF-κB via the carboxy-terminal activation region 1 and 2. Promoter deletion analysis indicated that two NF-κB binding sites are necessary for activation of the CD69 promoter. Electrophoretic mobility shift analysis demonstrated that LMP-1 activates both NF-κB binding sites in the CD69 promoter. This is the first report of the regulation of CD69 expression by LMP-1, and this novel finding may, thus, represent an important link between the EBV oncoprotein LMP-1 and its critical role in the development of EBV-associated diseases.

Modulation of IL-10-Specific B Cell MicroRNAs by Latent Membrane Protein 1 and Epstein-Barr Virus

Post-Transplant Lymphoproliferative Disorder (PTLD) is a potentially fatal side effect of the immunosuppressive agents used to prevent allograft rejection. PTLD is associated with Epstein-Barr Virus (EBV) infection and can result in malignant B cell lymphoma. Latent membrane protein 1 (LMP1), a viral mimic of the B cell costimulatory protein CD40, is the major oncogene of EBV and can usurp host cell pathways to promote cell survival. LMP1 activates the Akt/PI3K signaling pathway to drive IL-10 production, which is essential for the autonomous proliferation of EBV+ B cell lymphomas. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression to control cellular events such as differentiation, proliferation, and cell survival. As aberrant expression of microRNAs is found in a variety of human malignancies, our goal was to determine if microRNAs participate in LMP1-induced IL-10 production in PTLD-derived cell lymphomas. To first establish that EBV infection can modulate host cell microRNA, we used Taqman microRNA qPCR arrays to detect the microRNA expression profiles of six B cell tumor lines derived from patients with PTLD, two B cell lines generated by infection with the B95.8 strain of EBV obtained from a patient with mononucleosis, and one EBV-negative B cell line. Expression data was then normalized internally and to the EBV-negative B cell line. Our analysis showed that compared to the EBV-negative B cell line, B cells infected with the benign B95.8 strain of EBV had the altered expression (2-fold) of 294 microRNAs while the PTLD-derived EBV+ B cell lines had the altered expression of 224 microRNAs. Twenty-eight microRNAs were altered in both cases of EBV infection. To examine the specific role of LMP1 in host cell microRNA expression, we used an inducible system of LMP1 activation and expressed different LMP1 molecules in an EBV negative B cell lymphoma line. Activation of the B95.8 form of LMP1 modulated 62 cellular microRNAs and 142 microRNAs were modulated by all five of the PTLD-derived LMP1 molecules. Notably, only 28 microRNAs were modulated by both benign and tumor LMP1. The microRNAs aberrantly regulated by either EBV infection or LMP1 activation were next analyzed with target prediction software for potential binding to the 3″UTR of IL-10. MicroRNA-21, -106A, -106B, -194, -340, -374 and -374-5p fit these criteria and were tested for direct binding to the 3″UTR of IL-10 in luciferase reporter assays. The overexpression of microRNA-106A, -106B, -194, -374, -374-5p each negatively regulated the IL-10 3″UTR. Conversely, microRNA-21 overexpression positively modulated the 3″UTR of IL-10 while microRNA-340 overexpression had no effect. Together, these findings indicate that EBV infection and LMP1 activation induce host cell microRNAs that regulate production of the autocrine growth factor IL-10 in EBV+ B cell lymphomas. Thus, cellular microRNAs are a potential target for inhibiting the growth of PTLD-associated B cell lymphomas.

Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.

Latent membrane protein 1 of Epstein Barr Virus alters cellular microRNAs in B cell lymphomas (169.37)

Abstract Epstein-Barr Virus (EBV) infection is usually benign but immunodeficiency can lead to B cell lymphomas such as Post-Transplant Lymphoproliferative Disease (PTLD). PTLD is associated the expression of the EBV oncogene latent membrane protein 1 (LMP1). MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression. We hypothesized that LMP1 signaling in EBV+ B cell lymphomas can modulate cellular miRNA expression as a mechanism to promote viral persistence and/or host cell survival. Using an inducible, chimeric LMP1 signaling system we analyzed miR expression in a B cell lymphoma cell line after activation of LMP1 variants isolated from PTLD patients and from the B95.8 form obtained from a mononucleosis patient. The expression of 747 miRs was assayed by qPCR array and 323 miRs were detected. B95.8 LMP1 activation modulated 50 total miRs including miR503, linked to inhibition of proliferation in epithelial cells. The PTLD-derived LMP1 variants modulated 59 distinct miRs. These included pro-apoptotic miR29b which was down-regulated by PTLD-derived LMP1 variants but not by B95.8 LMP1. These data indicate that PTLD-derived forms of LMP1 uniquely alter cellular miR as compared to B95.8 LMP1. Thus, study of the functional properties of LMP1 variants may provide insight into mechanisms of lymphomagenesis.

Activation of the JAK/STAT Pathway in Epstein Barr Virus+-Associated Posttransplant Lymphoproliferative Disease: Role of Interferon-γ

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-kappaB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-kappaB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-kappaB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-kappaB factors in both B cells and epithelial cells. Exogenous expression of NF-kappaB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-kappaB activation and LMP1 expression.

The LMP1 oncogene of EBV activates PERK and the unfolded protein response to drive its own synthesis

The oncogene latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) without a ligand drives proliferation of EBV-infected B cells. Its levels vary in cells of clonal populations by more than 100-fold, which leads to multiple distinct activities of the oncogene. At intermediate levels it drives proliferation, and at high levels it inhibits general protein synthesis by inducing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). We have found that LMP1 activates PERK to induce phosphorylation of eIF2alpha, which upregulates activating transcription factor 4 (ATF4) expression. ATF4, in turn, transactivates LMP1's own promoter. LMP1 activates not only PERK but also inositol requiring kinase 1 (IRE1) and ATF6, 3 pathways of the unfolded protein response (UPR). Increasing expression levels of LMP1 induced a dose-dependent increase in IRE1 activity, as measured by its "splicing" of XBP-1. These infected B cells secrete immunoglobins independent of the levels of LMP1, indicating that only a threshold level of XBP-1 is required for the secretion. These findings indicate that LMP1's activation of the UPR is a normal event in a continuum of LMP1's expression that leads both to stimulatory and inhibitory functions and regulates the physiology of EBV-infected B cells in multiple, unexpected modes.

LMP1 signaling and activation of NF-κB in LMP1 transgenic mice

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.

Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis.

The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-kappaB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1's lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1's polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1's growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1's signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1's lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-kappaB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1's first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1's polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-kappaB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activity's being linked to individual membrane-spanning domains within LMP-1's polytopic transmembrane domain.

Functional analysis of different LMP1 proteins isolated from Epstein–Barr virus-positive carriers

The Epstein–Barr virus (EBV) is the causative agent of infectious mononucleosis and is implicated in the development of several human malignancies. Latent membrane protein 1 (LMP1), an EBV protein with known oncogenic properties, may be important in the pathogenesis of EBV-associated tumors, particularly nasopharyngeal carcinoma (NPC) and Hodgkin’s disease (HD). Several reports suggested that sequence variations in the LMP1 gene may define a more aggressive, geographically restricted EBV-genotype. Most mutations in the LMP1 gene described are located within the C-terminus of the protein. However, the effect of these mutations on the biological function of the protein remains widely unknown. Therefore, this study aimed in investigating whether mutations detected in LMP1 genes isolated from different EBV-positive carriers have an effect on the biological function of the protein. For this purpose the LMP1 genes were amplified by nested PCR from DNA out of bone marrow and peripheral blood lymphocytes and sequenced. Three functional assays were performed in order to evaluate the biological activity of the different isolates: activation of the transcription factors NF- κB and AP-1 as well as the anchorage independent growth of LMP1 transfected rat1 cells in soft agar. The results suggested that whereas differences in the activation of NF- κB through the various LMP1 isolates correlated tightly with their different expression levels, the outgrowth of transfected cells in soft agar did not and the transcription factor NF- κB therefore appeared not to be the major effector for the transformation of the rodent cell line rat1 by LMP1. The various LMP1-isolates also differed in their capacity in activating the transcription factor AP-1. We found no correlation between the transforming ability of the LMP1 isolates and activation of AP-1 suggesting that other so far uncharacterized domains also influence the transforming ability of the protein.

Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), activates NF-kappaB and triggers the transcription factor AP-1 via the c-Jun N-terminal kinase (JNK) cascade, but its specific contribution to B-cell immortalization has not been elucidated fully. To address the function of LMP1, we established B cell lines with a novel mini-EBV plasmid in which the LMP1 gene can be regulated at will without affecting the expression of other latent EBV genes. We demonstrate here that continuous expression of LMP1 is essential for the proliferation of EBV-immortalized B cells in vitro. Re-induction of LMP1 expression or activation of the cellular CD40 receptor both induce the JNK signaling cascade, activate the transcription factor NF-kappaB and stimulate proliferation of these B cells. Our findings strongly suggest that LMP1 mimics B-cell activation processes which are physiologically triggered by CD40-CD40 ligand signals. Since LMP1 acts in a ligand-independent manner, it replaces the T cell-derived activation signal to sustain indefinite B-cell proliferation.