Cathy McQuain

Carboxy terminal variants of Epstein-Barr virus-encoded latent membrane protein 1 during long-term human immunodeficiency virus infection : Reliable markers for individual strain identification

To assess the frequency and molecular polymorphism of malignancy-associated latent membrane protein 1 (LMP1) variants in human immunodeficiency virus type 1 (HIV-1) infection, 94 B-lymphoblastoid cell lines spontaneously derived from peripheral blood mononuclear cells (PBMC) and 30 PBMC samples at seroconversion and later (mean, 55 months) were analyzed by longitudinal comparative sequence analysis in 8 patients progressing to non-Hodgkins lymphoma (AIDS-NHL), 7 patients to opportunistic infections, and 2 patients with long-term asymptomatic HIV-1 infection. The sequence polymorphism in the C-terminus of LMP1 was characteristic for strains harbored by individual patients, with high fidelity for strain identification. In 14 of the 17 patients, two different but characteristic LMP1 variants were identified. At HIV seroconversion in 8 of 15 patients, a 30-bp deletion (LMP1Delta) was present. Though serial analysis revealed a shift to LMP1Delta in some individuals, statistical analysis of the cohort does not support the hypothesis that accumulation of LMP1Delta variants in PBMC accounts for their observed high incidence in AIDS-NHL.

The 30-bp Deletion Variant of Epstein-Barr Virus-Encoded Latent Membrane Protein-1 Prevails in Acute Infectious Mononucleosis

To assess the frequency of malignancy-associated 30-bp deletion variants of the latent membrane protein 1 (LMP-1) in benign conditions, a comparative sequence analysis was done using samples from 20 American children with acute infectious mononucleosis and 16 Swiss children with chronic tonsillar hyperplasia. The 30-bp deletion variant (LMP-1-del) was present in 66% of patients (12/20 with infectious mononucleosis and 12/16 with tonsillar hyperplasia). Two additional patients had a 3-bp deletion and an inframe insertion of 18 nucleotides, respectively. All but 1 isolate had numerous nonsilent point mutations. These data identify a hypervariable region within the C-terminus of LMP-1, in a domain required for maximal stimulation of NF-kappaB activity. These data demonstrate that LMP-1-del variants are frequent in acute infectious mononucleosis and tonsillar hyperplasia and identical to those observed in Epstein-Barr virus-associated AIDS-related lymphoma.

Natural 30 base pair and 69 base pair deletion variants of the LMP1 oncogene do stimulate NF-κB-mediated transcription

An increasing number of reports shows a link between the Epstein-Barr virus (EBV) and lymphoid neoplasia. The latent membrane protein 1 (LMP1) is likely to play a determinant role in this process since this EBV encoded protein has oncogenic properties and is usually expressed in EBV-associated lymphoproliferative diseases (LPD), except Burkitts lymphoma. We previously identified in LPD patients mutational hot spots and a 30 bp or 69 bp deletion in the LMP1 gene region coding for the C-terminal domain. These deletions are located in an area shown to be important for the activation of the transcription factor NF-κB. These findings lead us to test whether these natural deletion variants may have a functional effect. We measured the stimulation of their activity using a luciferase reporter plasmid containing NF-κB responsive elements. We tested the NF-κB inducing activity of four naturally occurring LMP1 deletion variants. Our results show that these deletion variants activate NF-κB to the same level as the wild-type form, indicating that the crucial residues for NF-κB activation are conserved among the variants isolated and lie within the last 32 amino acids of the C-terminal domain of the LMP1 oncogene.

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

Abstract 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.

Latent membrane protein 1 associated signaling pathways are important in tumor cells of Epstein-Barr virus negative Hodgkin's disease.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkins disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-κB mediated signaling at the step of IκB-α phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor IκB-α-NΔ54. However, dominant negative inhibition of NF-κB mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-κB mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Deletion Variants within the NF-kB Activation Domain of the LMP1 Oncogene in Acquired Immunodeficiency Syndrome-Related Large Cell Lymphomas, in Prelymphomas and Atypical Lymphoproliferations

The latent membrane protein 1 (LMP1) oncogene of Epstein Barr virus (EBV) is expressed in tumor cells of acquired immunodeficiency syndrome (AIDS) related lymphomas, HIV-negative, EBV-associated malignant lymphoproliferations, nasopharyngeal carcinoma, as well as in reactive immunoblasts of infectious mononucleosis. Naturally occurring LMP1 deletion variants (LMP1-del), characterized by clustered mutations and a distinct 30 base pair deletion within the carboxy terminal domain of LMP1, essential for maximal NF-kappaB stimulation, have been identified in the same conditions. These variants prevail in AIDS-related lymphomas, and are associated with clinically aggressive behaviour in HIV-negative lymphomas, and are frequent in prelymphomatous and reactive states. Functional studies showing a growth advantage of cells infected by these variants may explain the accumulation of LMP1-del in these entities. In the carboxy terminal NF-kappaB activation domain of LMP1, evidence of a hypervariable region close to the highly conserved 23 outermost amino acids essential for malignant transformation, may reflect the natural selection of growth promoting variants involved in signalling pathways. The prevalence of the same mutational pattern in AIDS-related lymphoma as well as in hyperplastic reactive states and prelymphomas supports the hypothesis that these variants confer a growth advantage manifested under impaired cellular immunity.

Sequence polymorphisms between latent membrane proteins LMP1 and LMP2A do not correlate in EBV‐associated reactive and malignant lympho‐proliferations

The latent membrane proteins LMP1 and LMP2A are co‐expressed in most malignancies associated with Epstein‐Barr virus (EBV). In contrast with the transforming LMP1 oncoprotein, LMP2A is expressed in lymphocytes of healthy EBV carriers and considered to maintain viral latency. Critical for these LMP2A functions are a transmembranous epitope recognized by specific cytotoxic T lymphocytes (CTLs) and the N‐terminal immunoreceptor tyrosine‐based activation motif (ITAM), blocking B‐cell receptor signaling. To characterize ITAM and CTL motifs of LMP2A and to correlate them with C‐terminal variants of LMP1 including the 30‐bp deletion variant (LMP1Δ), comparative sequence analysis was performed on 76 samples from patients with reactive and malignant lympho‐proliferation (infectious mononucleosis, n = 21; tonsillar hyperplasia, n = 16, chronic lympho‐proliferation, n = 9; Hodgkins disease, n = 8; Non‐Hodgkins lymphoma, n = 5; AIDS‐related large‐cell lymphoma, n = 17). The CTL motif was conserved in all but 2 cases (C426→S). The ITAM motif was characterized by strictly conserved YXXL sequences in all cases, with a sequence polymorphism in between. The B95.8 prototype was found in 17% (13/76) of cases, while in 72% a variant with 3 point mutations (166796 C→A, 166805 C→A, 166810 C→T) was detected; 11% had 1 or 2 of these mutations in addition to G→A at 166793. In the C terminus of LMP1, a hypervariable region including LMP1Δ was described in 61% of cases. There was no significant association of a particular LMP2A variant with either malignant phenotype or LMP1Δ, demonstrating that the functional domains of LMP2A are conserved and that the sequence polymorphisms in LMP1 and LMP2A are independent. Int. J. Cancer 81:371–375, 1999.

Selective outgrowth of a posttransplant B-immunoblastic lymphoma expressing a latent membrane protein-1 deletion variant.

BACKGROUND Posttransplant lymphoproliferative disorders are generally associated with Epstein-Barr virus (EBV) and are of B cell origin. We report the case of a B-immunoblastic lymphoma that developed in a pretransplantation EBV-seronegative woman 4 months after kidney transplant from her HLA-haploidentical brother. The patient successfully underwent immunotoxin therapy for lymphoma and has been in remission for 36 months. METHODS Latent EBV genomes were identified by polymerase chain reaction, and the purified amplification products were directly sequenced with [35S]dATP. RESULTS Molecular analysis of the latent membrane protein (LMP)1 oncogene of EBV, which was expressed in most tumor cells, revealed a 30-base pair deletion. No wild-type LMP1 sequences were found. Analysis of peripheral blood mononuclear cells from the EBV-seropositive donor showed the presence of both the LMP1 deletion variant and the wild-type sequence. The LMP1 deletion variant and the wild-type sequence were also identified within peripheral blood mononuclear cells of the EBV-seroconverted kidney recipient 20 months after lymphoma therapy. CONCLUSION This pattern is consistent with a natural growth advantage of B cells expressing the LMP1 deletion variant in the immunocompromised host.

Miliary tuberculosis in a patient with Epstein-Barr virus-associated angioimmunoblastic lymphadenopathy

A 74-year-old woman developed angioimmunoblastic lymphadenopathy (AILD) with involvement of intra-abdominal and retroperitoneal lymph nodes. Southern blot analysis showed germline configuration of the JH genes and an oligoclonal pattern of the TcRβ genes. The immunoblasts were of B-cell phenotype and often expressed the CD30 antigen and the latent membrane protein 1 (LMP1) oncogene. Six nonsilent point mutations were identified near the 3′ end of the LMP1 gene, leading to a cluster of six amino acid changes within a protein domain needed for maximal NF-κB stimulation. After a clinical remission of 8 months the patient relapsed with generalized lymphadenopathy and died secondary to tuberculosis. The oligoclonal rearrangements of the TcRβ genes may reflect an unsuccessful cellular immune response to Mycobacterium tuberculosis or an HLA-restricted T-cell response to B-immunoblasts expressing mutated viral antigens. A positive percutaneous tuberculin test observed 6 months prior to the onset of AILD is in favor of the first possibility.