Wild-type Latent Membrane Protein 1 Research Articles

Carboxyl terminal activating region 3 of latent membrane protein 1 encoded by the Epstein‑Barr virus regulates cell proliferation and protein expression in NP69 cells.

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein-Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232-351; amino acid residues including 232–351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69-LMP1Δ232-351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232-351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69-LMP1Δ232-351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2-fold change) in NP69-LMP1Δ232-351 cells compared with NP69-LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232-351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1-CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells.

Exosomal sorting of the viral oncoprotein LMP1 is restrained by TRAF2 association at signalling endosomes

The Epstein–Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1–TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1–TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1–TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1–TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that “signalling endosomes” in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

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.

Molecular Basis of Cytotoxicity of Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) in EBV Latency III B Cells: LMP1 Induces Type II Ligand-Independent Autoactivation of CD95/Fas with Caspase 8-Mediated Apoptosis

The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G(1) peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-kappaB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-kappaB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.

Proteome analysis of the transformation potential of the Epstein–Barr virus-encoded latent membrane protein 1 in nasopharyngeal epithelial cells NP69

Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.

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).

Epstein-Barr virus-encoded latent membrane protein 1 promotes stress-induced apoptosis upstream of caspase-2-dependent mitochondrial perturbation.

Previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) enhances etoposide-induced apoptosis in epithelial cells. Our study was undertaken to further dissect the modulation of tumor cell apoptosis by this viral protein. Using an inducible system of LMP1 expression in HeLa cells, we show herein that etoposide-triggered apoptosis, as evidenced by nuclear condensation and caspase-3 activation, is enhanced by LMP1. LMP1 also potentiates etoposide-induced processing and activation of caspase-2 in this model and enhances the dissipation of mitochondrial transmembrane potential and the release of cytochrome c in response to etoposide. Moreover, cisplatin-triggered activation of caspases 2 and 3 is potentiated upon expression of LMP1. A similar LMP1-mediated enhancement of cisplatin-induced caspase activation was seen upon stable transfection of wild-type LMP1 into the nasopharyngeal carcinoma cell line, TW03. Finally, using deletion mutants of LMP1 to determine the region of LMP1 required for apoptosis potentiation, we found that amino acids 350-386 (located within the CTAR2 domain) were responsible for sensitizing cells to cisplatin. We conclude that LMP1-dependent potentiation of stress-induced apoptosis occurs at an early step in the apoptosis cascade, upstream of the activation of caspase-2, and involves the C-terminal signaling domain of LMP1. These findings could have important ramifications for the treatment of EBV-associated malignancies of epithelial origin, including nasopharyngeal carcinoma.

A novel dominant-negative mutant form of Epstein-Barr virus latent membrane protein-1 (LMP1) selectively and differentially impairs LMP1 and TNF signaling pathways.

The latent membrane protein-1 (LMP1) is an integral membrane molecule expressed by Epstein-Barr virus (EBV) during viral latency and displays properties of a constitutively activated member of the TNF receptor family. LMP1 is required for B-cell or monocyte immortalization induced by EBV and is sufficient to transform rodent fibroblasts. Transforming potential of LMP1 is mediated by its cytoplasmic C-terminal domain, which activates various cellular signaling pathways including NFkappaB and JNK. In this report, we constructed mutants of LMP1 with preserved membrane spanning domain but mutated in the C-terminal domain and a second truncated C-terminal LMP1 fused to the enhanced green fluorescent protein. This latter mutant, termed LMP1-CT, impairs signaling by ectopic LMP1 as well as endogenous EBV-expressed wild-type (wt) LMP1. In contrast to dominant-negative mutants of LMP1 with preserved membrane spanning domains, LMP1-CT was unable to bind wt LMP1 to form an inactive complex. Its dominant-negative effects were due to binding and sequestration of LMP1 adapters TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. The effect was selective since LMP1-CT did not inhibit IL-1beta-induced signaling, whereas it impaired TNF-triggered NFkappaB and JNK signals without affecting TNF-induced apoptosis. In addition and in contrast to LMP1 constructs with membrane localization, LMP-CT did not display cytostatic properties in noninfected cells. Importantly, LMP1-CT inhibited survival induced by LMP1 in an EBV-transformed T-cell line expressing the type II viral latency commonly found in the majority of EBV-associated human tumors. These data demonstrate that LMP1-CT is a new tool to explore the differences between LMP1 and TNF signaling and may facilitate the design of molecules with potential therapeutic roles.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

LMP1, a viral relative of the TNF receptor family, signals principally from intracellular compartments.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.

LMP-1's transmembrane domains encode multiple functions required for LMP-1's efficient signaling.

The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-kappaB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-kappaB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Intracellular signaling molecules activated by Epstein-Barr virus for induction of interferon regulatory factor 7.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is the principal oncogenic protein in the EBV transformation process. LMP-1 induces the expression of interferon regulatory factor 7 (IRF-7) and activates IRF-7 protein by phosphorylation and nuclear translocation. LMP-1 is an integral membrane protein with two regions in its C terminus that initiate signaling processes, the C-terminal activator regions 1 (CTAR-1) and CTAR-2. Here, genetic analysis of LMP-1 has determined that the PXQXT motif that governs the interaction between LMP-1 CTAR-1 and tumor necrosis factor receptor-associated factors (TRAFs) is needed to induce the expression of IRF-7. Mutations in the PXQXT motif in CTAR-1 that disrupt the interaction between LMP-1 and TRAFs abolished the induction of IRF-7. Also, dominant-negative mutants of TRAFs inhibited the induction of IRF-7 by CTAR-1. The last three amino acids (YYD) of CTAR-2 are also important for the induction of IRF-7. When both PXQXT and YYD were mutated (LMP-DM), the LMP-1 mutant failed to induce IRF-7. Also, LMP-DM blocked the induction of IRF-7 by wild-type LMP-1. These data strongly suggest that both CTAR-1 and CTAR-2 of LMP-1 independently induce the expression of IRF-7. In addition, NF-kappaB is involved in the induction of IRF-7. A superrepressor of IkappaB (sr-IkappaB) could block the induction of IRF-7 by LMP-1, and overexpression of NF-kappaB (p65 plus p50) could induce the expression of IRF-7. In addition, we have found that human IRF-7 is a stable protein, and sodium butyrate, a modifier of chromatin structure, induces IRF-7.

Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells.

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IkappaBalpha(S32A/S36A), which is not phosphorylated and prevents NF-kappaB activation, with LMP1 showed that NF-kappaB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-kappaB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-kappaB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E(2) in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

The Epstein–Barr Virus Latent Membrane Protein 1 Induces Interleukin-10 in Burkitt's Lymphoma Cells but Not in Hodgkin's Cells Involving the p38/SAPK2 Pathway

Infection of B cells with Epstein–Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV–latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (ΔCTAR1) or 232 and 386 (ΔCTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NFκB activation by constitutive repressive IκB-α only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 μM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.

Mechanism of Action of a Novel Latent Membrane Protein-1 Dominant Negative

Latent membrane protein-1 (LMP1) is a signaling molecule expressed by Epstein-Barr virus during latency. LMP1 is essential for B-cell immortalization by Epstein-Barr virus and transforms rodent fibroblasts. It activates many distinct signaling pathways including the transcription factors NFkappaB and AP1. We have generated a mutant of LMP1 with four point mutations; amino acids 204, 206, and 208 were mutated to alanine, and amino acid 384 was mutated to glycine. This mutant, termed LMP1(AAAG), is not only unable to activate nuclear signaling pathways, but also inhibits signaling from wild type LMP1. We have demonstrated the effectiveness, selectivity, and mechanism of this inhibitory molecule. It inhibits LMP1-stimulated NFkappaB, STAT, and Jun transcriptional activity. It is selective, as it does not inhibit TNF or interleukin-2 signaling. We have demonstrated that it does not sequester the downstream signaling molecule, TRAF2, but instead binds LMP1 and interferes with its ability to bind TRAF2. This demonstrates the importance of the interplay between the signaling domains of LMP1 and the oligomeric structure of LMP1 for effective signaling. It identifies a tool that will be useful to probe LMP1 function in disease.

Latent membrane protein 1 of Epstein-Barr virus inhibits as well as stimulates gene expression.

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) functionally resembles a constitutively active, CD40-like receptor and contributes to the maintenance of proliferation of EBV-infected primary human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it binds TRAF, TRADD, and JAK molecules to activate NF-kappaB-, AP-1-, and STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a ligand to regulate its signaling. We have found that LMP-1, when expressed at physiologic levels, inhibits gene expression detectably. Higher levels of LMP-1 expression eventually inhibit both the steady-state level of RNA produced from a BamHI C promoter reporter and general cellular protein synthesis. These findings indicate that LMP-1 can limit its signaling and that this control is manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and JAK/STAT molecules is not required for this regulation. A derivative of LMP-1 that contains only its amino-terminal and membrane-spanning domains is sufficient to inhibit reporter activity when the reporter genes are expressed from the BamHI C and LMP-1 promoters. This same derivative of LMP-1 in parallel assays is sufficient to inhibit wild-type LMP-1's stimulation of NF-kappaB-dependent gene expression. We suggest that LMP-1 encodes stimulatory and inhibitory activities; the latter could limit signaling in the apparent absence of ligand-dependent down-regulation.

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.

Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2.

Nasopharyngeal carcinoma (NPC), which is closely associated with the Epstein-Barr virus (EBV), is a highly metastatic malignant tumor. An important activity in tumor invasion and metastasis is that of the 92-kDa type IV collagenase or gelatinase, matrix metalloproteinase 9 (MMP-9), which mediates the degradation of the basement membrane and extracellular matrix. The expression of MMP-9 has been shown to be enhanced by the EBV oncoprotein, latent membrane protein 1 (LMP-1). LMP-1, which is expressed in NPC, has two essential signaling domains within the carboxy terminus, termed C-terminal activation regions 1 (CTAR-1) and CTAR-2. This study reveals that either signaling domain can activate the MMP-9 promoter and induce MMP-9 activity; however, LMP-1 deletion mutants lacking either CTAR-1 or CTAR-2 had a decreased ability to induce MMP-9 expression. The deletion of both activation regions completely abolished the induction of MMP-9 activity, while the cotransfection of both the CTAR-1 and CTAR-2 deletion mutants restored MMP-9 activity to levels produced by wild-type LMP-1. The NF-kappaB and activator protein 1 (AP-1) binding sites in the MMP-9 promoter were essential for the activation of MMP-9 gene expression by both CTAR-1 and CTAR-2. The induction of MMP-9 expression by LMP-1 and both CTAR-1 and CTAR-2 mutants was blocked by the overexpression of IkappaB. The tumor necrosis factor receptor-associated factor (TRAF) pathway also contributed to the activation of the MMP-9 promoter as shown by the use of TRAF-2 and TRAF-3 dominant-negative constructs. These data indicate that the activation of both the NF-kappaB and AP-1 pathways by LMP-1, CTAR-1, and CTAR-2 is necessary for the activation of MMP-9 expression. In NPC, LMP-1 may contribute to invasiveness and metastasis through the induction of MMP-9 transcription and enzymatic activity.

Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression.

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.