Epstein-Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity.

A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.     

An Epstein-Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase.

Epstein-Barr virus (EBV) encodes two integral membrane proteins in latently infected growth-transformed cells. One of these, LMP1, can transform rodent fibroblasts and induce markers of B-lymphocyte activation. The second, LMP2, colocalizes with LMP1 in a constitutive patch in the EBV-transformed B-lymphocyte plasma membrane. The experiments reported here demonstrate that LMP2 may biochemically interact with LMP1 and that LMP2 closely associates with and is an important substrate for a B-lymphocyte tyrosine kinase in EBV-transformed B lymphocytes or in B-lymphoma cells in which LMP2 is expressed by gene transfer. LMP2 is also serine and threonine phosphorylated. LMP2 localizes to a peripheral membrane (presumably plasma membrane) patch in transfected B-lymphoma cells and colocalizes with much of the cellular tyrosine-phosphorylated proteins. LMP2 undergoes tyrosine phosphorylation in anti-LMP2 or antiphosphotyrosine immunoprecipitates from transfected B-lymphoma cells or EBV-transformed B lymphocytes. The first 167 of the 497 amino acids of LMP2 retain full ability to associate with and act as a substrate for a tyrosine kinase. A 70-kDa phosphotyrosine cell protein associates with LMP2 in transfected cells or in EBV-transformed B lymphocytes and could be a mediator of the effects of LMP2.     

An Epstein-Barr virus transforming protein associates with vimentin in lymphocytes.

The Epstein-Barr virus (EBV) latent infection membrane protein (LMP) is likely to be an important mediator of EBV-induced cell proliferation, since it is one of the few proteins encoded by the virus in latent infection and since production of this protein in Rat-1 cells results in their conversion to a fully transformed phenotype. LMP was previously noted to localize to patches at the cell periphery. In this paper we examine the basis of LMP patching in EBV-infected, transformed lymphocytes. Our data indicate that LMP is associated with the cytoskeletal protein vimentin. Although LMP is fully soluble in isotonic Triton X-100 buffer, only 50% of it is extracted from cells in this solution. The rest remains bound to the cytoskeleton. LMP undergoes phosphorylation, and phosphorylated LMP is preferentially associated with the cytoskeleton. As judged by both immunofluorescence and immunoelectron microscopy, the vimentin network in EBV-transformed lymphocytes or EBV-infected Burkitt tumor lymphocytes is abnormal. Vimentin and LMP often colocalize in a single patch near the plasma membrane. In response to Colcemid treatment of EBV-infected cells, vimentin reorganizes into perinuclear rings, as it does in uninfected cells. LMP is associated with these perinuclear rings. Vimentin (or a vimentin-associated protein) may be a transducer of an LMP transmembrane effect in lymphoproliferation.     

Epstein-Barr virus latent infection membrane protein increases vimentin expression in human B-cell lines.

Latent Epstein-Barr virus (EBV) infection activates B-lymphocyte proliferation through mechanisms which are partially known. One approach to further delineate these mechanisms is to identify cellular genes whose expression is augmented in cells latently infected with EBV. Since EBV-negative Burkitt's lymphoma cells can be grown in continuous culture and EBV can establish growth-altering latent infection in these cells, some effects of EBV on B-lymphocyte gene expression can be studied by using this in vitro system. Pursuing this latter approach, we have used cDNA cloning and subtractive hybridization to identify a gene whose expression is increased after EBV infection. This gene encodes the cytoskeletal protein vimentin. Latent infection of established EBV-negative Burkitt's lymphoma cell lines with the transforming EBV strain, B95-8, resulted in dramatic increases in vimentin mRNA and protein levels, while infection with the nontransforming P3HR1 strain failed to do so. Vimentin induction was reproduced by the expression of the single EBV gene which encodes the latent infection membrane protein (LMP). An amino-terminal LMP deletion mutant did not induce vimentin. These results are of particular interest in light of the transforming potential of LMP, as demonstrated in rodent fibroblasts, and the interaction between vimentin and LMP observed in immunofluorescent colocalization and cell fractionation studies.     

Functionally significant mutations in the Epstein-Barr virus <Emphasis Type="Italic">LMP1</Emphasis> gene and their role in activation of cell signaling pathways

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively activated analog of the tumor necrosis factor receptor TNF-R1. LMP1 serves as a viral oncogene able to transform human B-lymphocytes and rodent fibroblasts via activation of numerous cellular signal cascades. Two specific motifs within LMP1 are responsible for interaction of this viral protein with the receptor protein beta-TrCP/HOS SCF of the ubiquitin ligase E3 complex, playing an important role in degradation of numerous cellular proteins including NF-kappaB inhibitor IkappaBalpha. In this study, we demonstrate for the first time the importance of point mutations affecting HOS-recognizing motifs of LMP1 for activation of NF-kappaB, AP1, and PI3K/Akt signaling pathways. It has also been shown that rat fibroblast cell lines (Rat-1) expressing different HOS mutants of LMP1 produce different amounts of reactive nitrogen species. Our data confirm the hypothesis that point mutations in the C-terminal region of the LMP1 cytoplasmic domain can influence the transforming potential of the Epstein-Barr virus.     

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.     

LMP1 strain variants: biological and molecular properties

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-kappaB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med-, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-kappaB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.     

Unique signaling properties of CTAR1 in LMP1-mediated transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.     

A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1.

Recent cDNA cloning and sequencing of two Epstein-Barr virus (EBV)-specific mRNAs from latently infected cultures revealed that these RNAs are encoded across the fused terminal repeats of the viral genome and that they are likely to encode two nearly identical proteins with the same transmembrane domains. The smaller predicted protein (LMP2B) lacks 119 amino-terminal amino acids found in the larger one (LMP2A). To test whether these proteins are expressed in latently infected lymphocytes, antibodies to the LMP2 proteins were derived by immunizing rabbits with TrpE-LMP2A fusion proteins. Affinity-purified LMP2-specific antibodies recognized 54- and 40-kilodalton proteins, corresponding to LMP2A and LMP2B, in immunoblots of rodent fibroblasts stably transfected with eucaryotic expression plasmids containing either the LMP2A or LMP2B cDNA. Similar-size proteins were also identified in immunoblots of latently infected lymphocytes. LMP2A localized to membranes in cellular fractionation studies. In immunofluorescent studies, LMP2 localized in the plasma membrane of EBV-infected lymphocytes, with the majority of reactivity confined to the region of the LMP1 patch. This reactivity was detected in almost all lymphoblastoid cells latently infected with EBV.     

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23.

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)     

The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels.

A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Epstein-Barr virus. This gene encodes the latent membrane protein of Epstein-Barr virus. When the gene was expressed at high levels, it was toxic to all cell lines tested, which included six human B-lymphoid lines as well as BALB/3T3, 143/EBNA-1, and HEp-2 cells. The BNLF-1 gene was previously shown to induce anchorage-independent and tumorigenic growth in Rat-1 and BALB/3T3 cells. We demonstrate here that only those mutations in the BNLF-1 gene that score positively in the anchorage-independent growth assay were cytotoxic when expressed at high levels. It is therefore possible that the same activities of the latent membrane protein that are necessary to induce anchorage-independent growth of some rodent cell lines also confer toxicity to many cell lines when expressed at high levels.     

Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) has a short half-life (V. R. Baichwal and B. Sugden, J. Virol, 61:866-875, 1987; K.P. Mann and D. Thorley-Lawson, J. Virol, 61:2100-2108, 1987), is localized in patches in the membrane (D. Liebowitz, D. Wang, and E, Kieff, J. Virol, 58:233-237, 1986), and associates with the cytoskeleton in EBV-immortalized B lymphocytes (D. Liebowitz, R. Kopan, E. Fuchs, J. Sample, and E. Kieff, Mol. Cell. Biol. 7:2299-2308, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol. 61:2100-2108, 1987). Deletion mutants of LMP that are either positive or negative in the induction both of anchorage-independent growth of BALB/c 3T3 cells (V. R. Baichwal and B. Sugden, Oncogene 4:67-74, 1989) and of cytotoxicity in a variety of cells (W. Hammerschmidt, B. Sugden, and V. R. Baichwal, J. Virol. 63:2469-2475, 1989) have been studied to identify the biochemical properties of this protein that correlate with its effects on cell growth. Mutant LMP proteins that are metabolically stable, do not associate with the cytoskeleton, and exhibit a diffuse plasma membrane localization also do not induce anchorage-independent growth in rodent cells or cytotoxicity in B lymphoblastoid cells. In contrast, a mutant of LMP that is functionally identical to the wild-type protein has a half-life, membrane localization, and cytoskeletal association similar or identical to those of LMP. These results are consistent with the hypothesis that LMP's rapid turnover, association with the cytoskeleton, and patching in the membrane are required for it to affect cell growth.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

The latent membrane protein oncoprotein resembles growth factor receptors in the properties of its turnover.

The latent membrane protein (LMP) of Epstein-Barr virus functions as an oncogene in rodent cell lines (D. Wang, D. Liebowitz, and E. Kieff, Cell, 43: 831-840, 1985; V. R. Baichwal and B. Sugden, Oncogene, 2: 461-467, 1988) and, therefore, is likely to be essential for immortalization of human B-lymphocytes by Epstein-Barr virus. LMP has a short half-life in Epstein-Barr virus-infected B-lymphoblastoid cells (V. R. Baichwal and B. Sugden, J. Virol., 61: 866-875, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol., 61: 2100-2108, 1987) and in LMP-transformed rodent cell lines (V. R. Baichwal and B. Sugden, Oncogene 2: 461-467, 1988). The hypothesis that the turnover of LMP functions to down-regulate LMP activity has been tested by determining whether the turnover of LMP resembles that of several receptors for growth factors and neurotransmitters. The rapid turnover of LMP in transformed BALB/c 3T3 cells is blocked by cycloheximide, which indicates that turnover requires ongoing protein synthesis. Greater than 90% of newly synthesized LMP is present at the cell surface within 20 min of synthesis, and the detectable protein remains at this location for up to 6 h. If cells are grown in the presence of cycloheximide such that turnover of LMP is inhibited, an internalized pool of LMP can be detected; this observation indicates that turnover of LMP is likely to be preceded by internalization and that, once internalized, LMP is rapidly degraded. Also, this result indicates that the degradation of LMP, as opposed to its internalization, requires ongoing protein synthesis. The turnover of LMP and its biological activity (as assayed by cytotoxicity) are not regulated by factor(s) present only in serum, because the half-life of LMP in cells maintained in serum-free medium does not differ from that in the same cells grown in 5% calf serum. The rapid turnover, the requirement of protein synthesis for turnover, and the internalization of LMP are consistent with the functioning of this protein as a (ligand-dependent or independent) cell surface receptor.     

An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.