Tyrosine 112 of latent membrane protein 2A is essential for protein tyrosine kinase loading and regulation of Epstein-Barr virus latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

Explorers. Christopher Columbus and Leonardo da Vinci

Epstein-Barr virus (EBV) recently has been associated with Hodgkin's disease (HD) and the EBV genome was found in CD30-positive Reed-Sternberg cells. Therefore, tissue sections from 25 cases of HD, 35 cases of CD30-positive non-Hodgkin's lymphoma (NHL) (seven CD30-positive anaplastic large cell lymphomas [ALCLs] and 28 CD30-positive non-ALCLs), and 12 cases of CD30-negative NHL that previously had been screened for the presence of EBV by polymerase chain reaction and DNA in situ hybridization were studied by immunohistochemistry for the expression of the latent EBV proteins, latent membrane protein (LMP), and Epstein-Barr nuclear antigen-2 (EBNA-2). We also analyzed the expression of the B-cell activation molecule CD23 and the adhesion molecules LFA-1/CD11a and ICAM-1/CD54 because the upregulation of these molecules by LMP and/or EBNA-2 in vitro has been related to the EBV-induced lymphocyte growth. Latent membrane protein expression was found in Reed-Sternberg cells in nine of 25 cases (36%) of HD and in large, occasionally Reed-Sternberg-like tumor cells in six of 47 cases (12%) of NHL; these six tumors were CD30-positive, histologically high-grade NHL (one CD30-positive ALCL and five CD30-positive non-ALCLs). All the LMP-positive cases were also polymerase chain reaction EBV positive while LMP expression was not found in polymerase chain reaction EBV-negative HD and NHL. No staining for EBNA-2 was detected in our series. In view of the transforming potential of the LMP, these findings suggest that EBV may be associated with the development of some cases of HD and CD30-positive NHL. These findings also suggest a correlation between the expression of LMP and the detection of CD30 in tumor cells of HD and NHL. In contrast, no correlation was found between the expression of LMP and the detection of CD23, LFA-1/CD11a, and ICAM-1/CD54 in tumor cells of HD and NHL.     

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.     

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.     

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

In vivo growth of Epstein-Barr virus transformed B cells with mutations in latent membrane protein 2 (LMP2)

Epstein-Barr virus (EBV) causes infectious mononucleosis in adolescents and is associated with malignant B lymphocyte proliferation in AIDS patients, patients undergoing immune suppression for organ transplantation, and SCID mice. In vitro, EBV transformed, latently infected lymphoblastoid B cell lines (LCLs) contain EBV episomes and express nine virus encoded proteins. Six are nuclear proteins (EBNAs) and three are the integral membrane proteins, LMP1, LMP2A, and LMP2B. To determine if LMP2 was essential for in vivo growth, SCID mice were injected with LCLs containing wild-type EBV (LMP2+) or with LCLs transformed with EBV containing mutations in either LMP2A or LMP2B (LMP2-). SCID mice injected with the LMP2+ or LMP2- LCLs were monitored for tumor development, length of time to tumor development, and phenotypic characterization of the resulting tumors. No difference was observed in any of the above parameters between LMP2+ and LMP2- LCLs demonstrating that LMP2 is not essential for the in vivo growth of EBV transformed B lymphocytes in SCID mice.     

Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.     

Epstein-Barr virus latent membrane protein-1 oncogene deletion in post-transplantation lymphoproliferative disorders

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a multifunctional oncoprotein. A 30-bp deletion of the 3' end of the LMP1 gene (del-LMP1) has been identified in some EBV isolates. This deleted LMP1 gene encodes a protein, altered on the carboxy terminus, which is thought to have greater oncogenic potential than the wild type. Recently, it was suggested that del-LMP1 plays a role in the development of malignant lymphomas occurring in immunocompromised patients. To further elucidate the role of del-LMP1 in post-transplantation lymphoproliferative disorders (PT-LPDs) we analyzed 58 PT-LPD lesions from 36 heart and kidney organ transplant recipients. Overall, del-LMP1 was detected in 44% of the cases. Four plasmacytic hyperplasias (36%), eight polymorphic B-cell hyperplasias/polymorphic B-cell lymphomas (38%), and five malignant lymphomas/multiple myelomas (71%) exhibited del-LMP1. Two of the three patients displaying disease progression showed wild-type LMP1 gene (w-LMP1) and one showed del-LMP1. LMP1 status remained the same in all three patients during disease progression. In patients undergoing biopsy of multiple separate PT-LPD lesions representing different clonal lymphoid proliferations, LMP1 status was the same in all of the lesions in each patient. Furthermore, although the polyclonal lesions harbor multiple EBV infectious events, they either showed w- or del-LMP1 but not both. Analysis of the tissues without an apparent PT-LPD (peripheral blood, bone marrow, or colon) revealed EBV and LMP1 type identical to that found in the lesions. In conclusion, the presence or absence of del-LMP1 in PT-LPDs does not correlate with the histopathological category or the malignant nature of the lymphoid proliferation. LMP1 status does not change during disease progression and is the same within multiple lesions occurring in the same patient regardless of their clonal relationship. These findings suggest that 1) EBV infection in patients with PT-LPDs occurs with a w- or del-LMP1-type EBV isolate and does not change once a patient acquires the virus and 2) the infection is an early event in the development of PT-LPDs and transformation is induced regardless of the type of LMP1.     

Lung cancer: intermittent irradiation synchronized with respiratory motion--results of a pilot study

Pyothorax-associated lymphoma (PAL) is a newly-described entity developing several decades after artificial pneumothorax treatment for pulmonary or pleural tuberculosis. It is known to be associated with Epstein-Barr virus (EBV) with constant expression of the two latent membrane proteins: latent membrane protein (LMP)-1 and EBV-associated nuclear antigen (EBNA)-2. We are reporting three new cases of PAL. All of the tumours were of B-cell lineage and classified as large-cell diffuse lymphomas according to the International Working Formulation for the Classification of Lymphomas. The EBV genome was detected in two of the cases with LMP-1 and EBNA-2 expression. No EBV could be detected in the third case suggesting that different mechanisms may be involved in the pathogenesis of the disease. Body cavity-based high grade lymphomas (BCBL) represent a new disease, developing mainly in human immunodeficiency virus (HIV) infected patients: the tumoural cells often contain both human herpes virus (HHV)-8 (or Kaposi's sarcoma herpes virus) and EBV genomes, suggesting that these viruses might co-operate in the pathogenesis of the disease. The pleural location and the association of EBV have led to speculation that PAL could also be related to HHV-8 infection. However, no HHV-8 genome could be detected in any of the 14 tested cases already reported in the literature nor in the two cases we studied (one EBV-positive and one EBV-negative), suggesting that PAL and BCBL are two different entities.     

Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes

Epstein-Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed alphav integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.     

Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines

B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.