High expression of latent membrane protein 1 of Epstein-Barr virus and BCL-2 oncoprotein in acquired immunodeficiency syndrome-related primary brain lymphomas

Nearly all primary brain lymphomas in acquired immunodeficiency syndrome (AIDS) patients are associated withEpstein-Barr virus (EBV). The role of EBV in lymphomagenesis is not totally elucidated. One possible mechanism is the overexpression of the BCL-2 oncoprotein, because the latent membrane protein 1 (LMP1) has been reported to transactivate the bcl-2 gene in vitro. To study the interrelationship beetween LMP1 and BCL-2 in vivo, we have analyzed and compared their expression in 11 AIDS-related primary brain lymphomas and 57 AIDS- related systemic lymphomas by immunoperoxidase technique on frozen sections. In AIDS-related primary brain lymphoma, LMP1 and BCL-2 were expressed in all cases but 1. All positive cases exhibited morphologic immunoblastic features. In contrast, the only negative case was histologically close to Burkitt's lymphoma. In systemic lymphomas, LMP1 was expressed in 21 cases, whereas BCL-2 was positive in only 3 cases, all of which were extranodal. These results indicate that, in addition to the histologic type, the role of EBV genes and BCL-2 expression in lymphomatous cells differ as a function of their localization. In AIDS- related primary brain lymphomas, this correlation between LMP1 and BCL- 2 overexpression may have a major implication in lymphomagenesis.     

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.     

Activation of alternative NF-kappa B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein (vFLIP)

The Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) has been linked to KS and primary effusion lymphoma (PEL) in immunocompromised individuals. We report that PEL cell lines have constitutive active alternative NF-kappa B pathway and demonstrate high-level expression of NF-kappa B2/p100 precursor and its processed subunit p52. To elucidate the mechanism of activation of the alternative NF-kappa B pathway in PEL cells, we have investigated the role of KSHV-encoded viral Fas-associated death domain-like IL- beta 1-converting enzyme inhibitory protein (vFLIP) K13. We demonstrate that stable expression of K13, but not other FLIPs, in a variety of cell lines constitutively up-regulates p100/NF-kappa B2 expression and leads to its processing into the p52 subunit. K13-induced up-regulation and processing of p100 critically depends on the I kappa B kinase (IKK)alpha/IKK1 subunit of the IKK complex, whereas IKK beta/IKK2, receptor-interacting protein, and NF-kappa B-inducing kinase are dispensable for this process. Silencing of endogenous K13 expression by siRNA inhibits p100 processing and cellular proliferation. Our results demonstrate for the first time, to our knowledge, that KSHV vFLIP K13 is required for the growth and proliferation of PEL cells and alternative NF-kappa B pathway plays a key role in this process. Therapeutic agents targeting the alternative NF-kappa B pathway may have a role in the treatment of KSHV-associated lymphomas.     

Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat.

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-regulated gene expression is stimulated independently by the cellular trans-activator NF-kappa B and the viral protein Tat. Noncytotoxic concentrations of the drug pentoxifylline (PTX) inhibited interaction of NF-kappa B with its motif and the stimulation of HIV-1 LTR-driven gene expression in Jurkat cells. Tat protein (from a cotransfected Tat-expression vector) also induced activation of HIV-1 LTR-driven gene expression. This activation was unaffected by PTX when NF-kappa B sites in the HIV-1 LTR were mutated, suggesting that this drug does not directly influence Tat function, which, however, was inhibited by the Tat-inhibitor Ro 24-7429. Transient reporter gene expression regulated by HIV-1 LTR with wild-type NF-kappa B motifs in the presence of Tat protein was 10- to 60-fold higher than in the presence of either of the trans-activators alone, demonstrating superactivation of HIV-1 LTR by the concerted action of both the trans-activators. Treatment of cells with either PTX or Ro 24-7429 inhibited this superactivation of the HIV-1 LTR. The inhibitory effect of these two drugs in combination, at concentrations that alone did not significantly influence viral promoter activity, was far more than additive. A cooperative action of PTX (NF-kappa B inhibitor) and Ro 24-7429 (Tat inhibitor) on HIV-1 LTR-regulated gene expression is suggested. Concentrations of the drugs that induced maximum inhibition of HIV-1 LTR through their cooperative action are far below cytotoxic levels. Thus, the combination of these two inhibitors could be very effective for anti-HIV therapy.     

Epstein-Barr virus-associated persistent polyclonal B-cell lymphocytosis with a distinct 69-base pair deletion in the LMP1 oncogene

 Epstein-Barr virus (EBV) genomes have been detected in peripheral blood lymphocytes (PBL) of patients with persistent polyclonal B-cell lymphocytosis (PPBL). This is consistent with the hypothesis that latent EBV infection is involved in the pathogenesis of this disorder. Two EBV-encoded proteins expressed in viral latency are the latent membrane proteins 1 and 2A (LMP1 and LMP2A). We have studied the LMP1 oncogene and the LMP2A gene in a female patient with PPBL and her five siblings. A cell line derived from peripheral blood lymphocytes (PBL) of the patient was also analyzed. A distinct 69-base pair deletion was identified within the carboxy terminal NF-κB activation domain of the LMP1 oncogene in PBL of the patient and in the cell line, whereas none of the siblings harbored this deletion. The tyrosine-signaling motif and the HLA A2.1 epitope of the LMP2A gene were wild type in the patient and all siblings. The presence of a 69-base pair deletion variant of the LMP1 oncogene within the lymphocytes of a PPBL patient but absence of this deletion variant in the unaffected siblings suggests a direct implication of altered LMP1 oncoprotein-dependent function in the pathogenesis of PPBL. Received: 26 August 1996 / Accepted: 24 October 1996     

IRF7 activation by Epstein–Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3

Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1), a constitutively aggregated and activated pseudoreceptor, activates IFN regulatory factor 7 (IRF7) through RIP1. We now report that the LMP1 cytoplasmic carboxyl terminal amino acids 379–386 bound IRF7 and activated IRF7. IRF7 activation required TRAF6 and RIP1, but not TRAF2 or TRAF3. LMP1 Y₃₈₄YD₃₈₆, which are required for TRADD and RIP1 binding and for NF-κB activation, were not required for IRF7 binding, but were required for IRF7 activation, implicating signaling through TRADD and RIP1 in IRF7 activation. Association with active LMP1 signaling complexes was also critical for IRF7 activation because (i) a dominant-negative IRF7 bound to LMP1, blocked IRF7 association and activation, but did not inhibit LMP1 induced NF-κB or TBK1 or Sendai virus-mediated IFN stimulated response element activation; and (ii) two different LMP1 transmembrane domain mutants, which fail to aggregate, each bound IRF7 and prevented LMP1 from binding and activating IRF7 in the same cell, but did not prevent NF-κB activation. Thus, efficient IRF7 activation required association with LMP1 CTAR2 in proximity to LMP1 CTAR2 mediated kinase activation sites.     

NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene promoter underlies self-perpetuation of human immunodeficiency virus transcription in monocytic cells.

The molecular mechanisms underlying the sustained nuclear translocation of NF-kappa B observed in U937 monocytic cells chronically infected with human immunodeficiency virus (HIV) were studied. The activity of the promoter regulating the synthesis of the p105 precursor of the NF-kappa B p50 subunit was enhanced in these cells. Deletions in this promoter indicated that this upregulation was mediated through the NF-kappa B- but not the AP-1-binding motif, by bona fide p50/p65 heterodimers. Analysis of cytosolic extracts indicated that NF-kappa B levels were increased in HIV-infected cells. In contrast to the transient NF-kappa B activation induced by phorbol ester, the permanent NF-kappa B translocation induced by HIV infection was not dependent on PKC isoenzymes alpha and beta as shown by the use of a specific inhibitor (GF 109203X). These observations indicate that during chronic HIV infection of U937 cells, continuous NF-kappa B (p50/p65) translocation results in p105 promoter upregulation with subsequent cytosolic NF-kappa B accumulation, ready for further translocation. This HIV-mediated mechanism results in a self-perpetuating loop of NF-kappa B production.     

Epstein-Barr virus infection and associated products (LMP,EBNA2, vIL-10) in nodal non-Hodgkin's lymphoma of human immunodeficiency virus-negative Japanese

Sixty cases of B-cell nodal non-Hodgkin's malignant lymphoma (B-ML), and 46 cases of T-cell nodal lymphoma (T-ML) were surveyed for Epstein-Barr virus (EBV) genomes, RNA, and associated proteins. We used a Southern blot analysis, polymerase chain reaction (PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the presence of EBV. We performed an immunohistochemical study on EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 (EBNA-2), latent membrane protein (LMP), and viral interleukin-10 (vIL-10). In addition, we also analyzed the terminal repetitive sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin's lymphomas were grouped into three types by number of EBV-infected cells: I) almost all lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed an EBV presence; and III) only a few cells showed such a presence, which was probably due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infected cells were found; 7 were type I, 1 was type II, and 17 were type III. In 27 of 46 T-MLs, EBV-infected cells were found; no cases were type I, 5 cases were type II, and 22 cases were type III. Seven B-MLs and 3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-2/LMP-positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was detected in almost all lymphoma cells; In T-MLs, however, LMP was found in only a small portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In summary, it was thus speculated that EBV infection was associated with the various states of lymphomagenesis. © 1996 Wiley-Liss, Inc.     

Lymphomas of mucosal-associated lymphoid tissue in common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency disease characterized by low serum immunoglobulins IgG, IgA, and usually IgM. The central immune deficiency is impaired secretion of immunoglobulins and lack of antibody production; however, T cell dysfunction and a variety of inflammatory complications suggest global immune dysregulation. A number of reports have documented the association of primary immunodeficiency diseases with the development of non-Hodgkin's lymphoma (NHL). In CVID, the risk has been estimated to lie between 1.4% and 7%. As for NHL arising in other immunodeficiency states, the lymphomas in CVID are extranodal and are usually B cell in type. Of 22 B cell lymphomas that have appeared over a period of 25 years in a cohort of subjects with CVID, five lymphomas, appearing in more recently studied subjects, that arose in mucosal sites would be classified as mucosa-associated lymphoid tissue (MALT) lymphomas. MALT lymphomas are low-grade B cell lymphomas that result from a proliferation of neoplastic marginal-zone related cells of lymphoid tissue and tend to occur in organs that have acquired lymphoid tissue due to long-term infectious or autoimmune stimulation. Lymphomas of this kind have not been described in patients with congenital immunodeficiency, although chronic mucosal antigen stimulation is an integral part of these immune deficiency states.     

Frequency of a 30-base pair deletion in the latent membrane protein-1 gene in Epstein-Barr virus-associated lymphomas in Japan

The association of Epstein-Barr virus (EBV) with various lymphoid malignancies has been reported. The precise pathogenesis of EBV in malignancies has not yet been elucidated. Latent membrane protein-1 (LMP-1) and Epstein-Barr nuclear antigen-2 (EBNA-2) genes are suspected to be tumorigenic genes. Previous studies suggest that a deletion within the LMP-1 gene may increase the oncogenic potential of EBV. In this study, we analyzed the sequence within the carboxy terminal end of the LMP-1 gene in paraffin-embedded specimens from T-cell lymphomas, Hodgkin's disease (HD), and the buffy coat of peripheral blood from healthy individuals in Japan. Polymerase chain reaction (PCR) was performed using primers spanning the carboxy terminal region of the LMP-1 gene, and sequence analysis was performed to show the exact location of the deletion. The PCR product of the Raji cell line was 161 base pairs (bp), and the LMP-1 gene with deletion was 30 bp shorter in a direct sequence of PCR products. The 30-bp deletion was located in position 168285-168256 of the Raji cell. A deletion within the LMP-1 gene was found in 4 of 25 cases (16%) of EBV-positive T-cell lymphomas, 4 of 10 cases (40%) of EBV-positive HD cases, and 2 of 13 specimens (15%) with amplified PCR products from 49 healthy individuals. The incidence of the 30-bp deletion within the LMP-1 gene in HD was comparable to that of subjects in the United States and Brazil, but the deletion was not found in a high proportion of EBV-positive T-cell lymphoid malignancies. No statistical significance was found regarding the clinical outcome between patients with a deletion within the LMP-1 gene and patients with wild-type LMP. This deletion cannot be considered as simply causing the pathogenesis of EBV-associated lymphoid malignancies in Japan.