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.     

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.     

Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3.

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizing activity of EBV in primary, human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it influences signaling pathways of infected cells. LMP-1 has been found to associate with members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. As with LMP-1, the TRAF molecules have been shown to participate in cell signaling pathways. We have characterized and mapped in detail a region of LMP-1 that associates with TRAF1, TRAF2, and TRAF3. TRAF3 alone associates with LMP-1 in a yeast two-hybrid assay, whereas all three TRAF molecules associate with LMP-1 under various conditions when they are assayed in extracts of human cells. TRAF1, TRAF2, and TRAF3 appear to associate independently with LMP-1 but bind an overlapping target site. TRAF3 associates with LMP-1 most avidly and can compete with TRAF1 and TRAF2 for binding to LMP-1. TRAF2 associates with truncated derivatives of the carboxy terminus of LMP-1 more efficiently than with the intact terminus, indicating that LMP-1's conformation may regulate its association with TRAF2. Finally, point mutations that decrease LMP-1's association with the three TRAF molecules to 3 to 20% of wild-type levels do not detectably affect otherwise intact LMP-1's induction of NF-kappaB activity. Therefore, these associations are not necessary for the majority of intact LMP-1's induction of this signaling pathway.     

A membrane leucine heptad contributes to trafficking, signaling, and transformation by latent membrane protein 1

Latent membrane protein 1 (LMP1) of Epstein Barr virus (EBV) is important for maintaining proliferation of EBV-infected B cells. LMP1, unlike its cellular counterpart, CD40, signals without a ligand and is largely internal to the plasma membrane. In order to understand how LMP1 initiates its ligand-independent signaling, we focused on a leucine heptad in LMP1's first membrane-spanning domain that was shown to be necessary for LMP1's signaling through NF-kappaB. LZ1EBV, a recombinant EBV genetically altered to express LZ1, a derivative of LMP1 in which a leucine heptad was replaced with alanines, transformed B cells with 56% of wild-type (wt) EBV's efficiency, demonstrating the importance of this heptad. To elucidate the mechanism by which this domain contributes to the functions of LMP1, the properties of the wt and LZ1 were compared in transfected cells. LZ1 failed to home to lipid rafts as efficiently as did wt LMP1. The distribution of tagged derivatives of LZ1 also differed from that of wt LMP1 in transfected cells. LZ1's defect in homing to lipid rafts and altered trafficking likely underlie the defect in transformation of LZ1EBV. While the third and fourth membrane-spanning domains of LMP1 foster its trafficking to the Golgi, the leucine heptad within the first membrane-spanning domain contributes to its trafficking, particularly to internal rafts. B cells that are successfully transformed by LZ1EBV have the same average number of viral genomes and the same fraction of cells with capped LZ1 at the cell surface but express 50% more of the LZ1 allele than wt infected cells.     

The late lytic LMP-1 protein of Epstein-Barr virus can negatively regulate LMP-1 signaling

The BNLF-1 open reading frame of Epstein-Barr virus (EBV) encodes two related proteins, latent membrane protein-1 (LMP-1) and lytic LMP-1 (lyLMP-1). LMP-1 is a latent protein required for immortalization of human B cells by EBV, whereas lyLMP-1 is expressed during the lytic cycle and is found in the EBV virion. We show here that, in contrast to LMP-1, lyLMP-1 is stable, with a half-life of >20 h in tetradecanoyl phorbol acetate- and butyrate-treated B95-8 cells. Although lyLMP-1 itself has negligible effects on NF-kappaB activity, it inhibits NF-kappaB activation by LMP-1 in a dose-dependent manner. The lyLMP-1 protein does not oligomerize with LMP-1, and the negative effect of lyLMP-1 on NF-kappaB activation by LMP-1 does not result from lyLMP-1-mediated disruption of LMP-1 oligomers. Modulation of LMP-1-activated signaling pathways is the first identified biological activity associated with lyLMP-1, and this activity may contribute to the progression of EBV's lytic cycle.     

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.     

Oncogenic activity of Epstein-Barr virus latent membrane protein 1 (LMP-1) is down-regulated by lytic LMP-1

The Epstein-Barr virus (EBV) is an oncogenic human herpesvirus. EBV latent membrane protein 1 (LMP-1) is a viral oncogene that manifests its oncogenic phenotype through activation of cellular signaling pathways involved in cell growth, survival, differentiation, and transformation. Lytic LMP-1 (lyLMP-1) is a related EBV gene without oncogenic properties. The lyLMP-1 gene is found in 60% of the EBV strains circulating in nature, but it is not found in EBV strains associated with nasopharyngeal carcinoma. We recently demonstrated that lyLMP-1 down-regulates the half-life of LMP-1 in epithelial cells. Therefore in this study, we tested the hypothesis that lyLMP-1 concomitantly down-regulates LMP-1 oncogenic activity. The results demonstrated that lyLMP-1 inhibits LMP-1-mediated intracellular signaling activation, epithelial cell growth and survival, and fibroblast cell transformation in a dose-dependent manner. Lytic LMP-1 manifested this effect through the promotion of LMP-1 degradation and a reduction in the expressed quantity of LMP-1. Thus, lyLMP-1 functions as a posttranslational negative regulator of LMP-1 oncogenesis. These results support a model of EBV-associated epithelial oncogenesis in which lyLMP-1 may act in vivo to reduce the risk of LMP-1-mediated transformation and is therefore subjected to negative selection in nasopharyngeal carcinoma pathogenesis.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

PRA1 promotes the intracellular trafficking and NF-kappaB signaling of EBV latent membrane protein 1

Latent membrane protein 1 (LMP1), which is an Epstein-Barr virus (EBV)-encoded oncoprotein, induces nuclear factor-kappa B (NF-kappaB) signaling by mimicking the tumor necrosis factor receptor (TNFR). LMP1 signals primarily from intracellular compartments in a ligand-independent manner. Here, we identify a new LMP1-interacting molecule, prenylated Rab acceptor 1 (PRA1), which interacts with LMP1 for the first time through LMP1's transmembrane domain, and show that PRA1 is involved in intracellular LMP1 trafficking and LMP1-induced NF-kappaB activity. Immunofluorescence and biochemical analyses revealed that LMP1 physically interacted with PRA1 at the Golgi apparatus, and the colocalization of LMP1 and PRA1 to the Golgi was sensitive to nocodazole and brefeldin A. Coexpression of a PRA1 export mutant or knockdown of PRA1 led to redistribution of LMP1 and its associated signaling molecules to the endoplasmic reticulum and subsequent impairment of LMP1-induced NF-kappaB activation, but had no effect on CD40- and TNFR1-mediated signaling or the functional integrity of the Golgi apparatus. These novel findings provide important new insights into LMP1, and identify an unexpected new role for PRA1 in cellular signaling.     

All three domains of the Epstein-Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts.

LMP-1, the Epstein-Barr virus latent membrane protein 1, is the only protein encoded by the virus that has been shown to have the properties of a transforming oncogene in rodent fibroblasts such as Rat-1 cells. LMP-1 is phosphorylated and proteolytically cleaved in Rat-1 cells in a manner similar to that seen in human lymphocytes. In this study, we demonstrate that all three major domains of LMP-1 (N-terminal, transmembrane, and C-terminal domains) are required for the ability to transform Rat-1 cells in culture, as assayed by loss of contact inhibition. This study is the first demonstration of a functional role for the C-terminal domain of LMP-1. Our analysis suggests that there are at least three distinct regions of the C terminus involved in signalling. Amino acids 306 to 334, which generate a toxic signal in the absence of amino acids 334 to 364, and the last 23 amino acids, 364 to 386, are essential for transformation. Biochemical analysis of the LMP-1 mutants with the three domains deleted indicate that the mutant N-terminal with the domain deleted is phosphorylated normally but is inefficiently cleaved compared with the wild-type LMP-1. The mutant with the transmembrane domain deleted is also phosphorylated but is not cleaved, showing that phosphorylation of LMP-1 does not require membrane association. The nontransforming mutant with the C-terminal domain deleted that lacks the last 23 amino acids is phosphorylated and cleaved. Therefore, these processing events alone are insufficient to generate a transforming signal.     

Epstein-Barr virus latent membrane protein 1 (LMP-1) half-life in epithelial cells is down-regulated by lytic LMP-1

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.     

Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.