Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein

Chen, Haifeng; Kevan-Jah, Sarah; Suentzenich, Karl-Otto; Grässer, Friedrich A.; Mueller-Lantzsch, N.

doi:10.1016/0042-6822(92)91196-2

Cited by 21 publications

(16 citation statements)

References 32 publications

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“…25 In our view, therefore, sequence divergence is unlikely to have led to a significant underestimate of LMP-specific immunity. The present data indicating that the LMPs are only weakly immunogenic accord with the finding that the LMPs only induce detectable humoral responses in rare circumstances; thus anti-LMP1 antibodies have been detected in a subset of patients with HD or NPC but not in most healthy carriers, 23,31,32 while anti-LMP2 antibodies have only been described in NPC patients. 23,33,34 Furthermore, our recent studies have shown that LMP1 and LMP2 are also a poor source of epitopes for the CD4 ϩ T cell response.…”

Section: Discussionsupporting

confidence: 88%

Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

Meij

Leen

Rickinson

et al. 2002

Intl Journal of Cancer

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Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expressionprofiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8 ؉ T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFN␥-ELISPOT assays of interferon-␥ release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4 ؉ or CD8 ؉ T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8 ؉ T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1-and 5 new LMP2-derived CD8 ؉ epitopes were identified. In most donors LMP1-and LMP2-specific CD8 ؉ precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8 ؉ memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use. © 2002 Wiley-Liss, Inc. Key words: CTL; LMP1; LMP2; Epstein-Barr virusEpstein-Barr virus (EBV) can induce fatal B lymphoproliferative lesions in immunocompromised patients and is etiologically linked to several malignancies arising in the immunocompetent host. Examples of the latter include all cases of endemic Burkitt's lymphoma (BL), all undifferentiated nasopharyngeal carcinomas (NPC), 10% of gastric carcinomas, certain T-/NK-cell lymphomas and 40 -90% of Hodgkin's Disease (HD) cases. 1 EBV is transcriptionally active in the tumor cells but expresses only a restricted set of EBV-encoded proteins; expression patterns range from the Epstein-Barr nuclear antigen 1 (EBNA1) only in BL through to EBNA1 plus high levels of both latent membrane proteins 1 and 2 (LMP1, -2) in HD. 2-4 EBV-associated carcinomas are intermediate in that EBNA1 and LMP2 appear to be expressed, with or without LMP1. 5 Interestingly many EBV-positive carcinomas also show detectable transcription of a gene, BARF1, which is otherwise thought to be associated only with the virus lytic cycle. 6,7 Given the presence of at least some viral antigens in tumor cells, EBV-positive malignancies are potential candidates for immunotherapy. Because endogenously expressed EBNA1 is protected from proteasomal processing and therefore escapes cytotoxic T lymphocyte (CTL) recognition, 8 the prime targets for therapeutic responses in the context of HD and NPC are LMP1 and LMP2. Both are multiple mem...

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Section: Discussionsupporting

confidence: 88%

Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

Meij

Leen

Rickinson

et al. 2002

Intl Journal of Cancer

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“…A 1.95-kb fragment containing a full-length LMP1 gene (pLMP1 [6]) was removed and inserted into the BamHI site of pcDNA3 to give pcDNA3-LMP1. The LMP1 fragment then was isolated with HindIII and EcoRV digests and cloned into HindIII and ClaI sites of the retroviral vector pLNSX (31) to generate pLNSX-LMP1.…”

Section: Methodsmentioning

confidence: 99%

LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene

Yang¹,

Sham²,

Ng³

et al. 2000

J Virol

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The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-Epstein-Barr virus (EBV) is a human gammaherpesvirus commonly carried in the majority of the human population. It is the causative agent for proliferative diseases such as infectious mononucleosis and oral hairy leukoplakia in AIDS patients. It is also implicated in a variety of human malignancies that include Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, nasal T-cell lymphoma, and immunoblastic lymphomas in posttransplant and AIDS patients (39). The virus often adopts latent forms in EBV-associated cancers. In nasopharyngeal carcinoma and Hodgkin's lymphoma, type II latency gene expression was observed. During this type II latency, only three viral proteins are routinely detected: EBV nuclear antigen 1 (EBNA1) and two latent membrane proteins, LMP1 and LMP2. Among the three EBV genes, the LMP1 gene is the only one implicated in cell immortalization and transformation. It is one of the viral genes required for B-cell immortalization, together with the EBNA2, EBNA3a, and EBNA3c genes (8,15,22,23,48). In addition, LMP1 has been shown to induce the transformation of certain established rodent fibroblast cell lines, including Rat-1 and BALB/c 3T3 (2, 33, 49). Furthermore, LMP1 induces the morphological transformation of the RHEK-1 cell line (12) and the tumorigenicity of epithelial cell lines in severe combined immunodeficient mice (18, 36).Although EBV is an important oncogenic virus in human, the LMP1 genes appeared to act differently from oncogenic genes of other DNA oncogenic viruses, including simian virus 40 (SV40) large T antigen, adenovirus E1A and E1B, and human papillomavirus (HPV) E6 and E7. There is little evidence suggesting that the LMP1 gene can function similarly to these viral oncogenes or even a cellular oncogene such as ras or myc. Though LMP1 transforms several immortalized rodent fibroblast cell lines and participates in B-cell immortalization, a single LMP1 gene has not been shown to induce continuous proliferation of any primary cell culture. Similarly, although it cooperates with a number of EBV genes in inducing B-cell proliferation, there is no report on the cooperativity of the LMP1 gene with another cellular oncogene in transforming primary cells.Protein sequence analysis of LMP1 revealed at least three domains: an amino-terminal cytoplasmic domain of 20 amino acid residues, a six-transmembrane domain of 185 amino acid residues, and a carboxy-terminal cytoplasmic domain of 200 amino acid residues (24). The six-transmembrane domain was shown to be required to form cytoplasmic membrane patches. The oligomerization of LMP1 in the cytoplasmic membrane may mimic that of other membrane receptor molecules induced by ligand-receptor interactions, resulting in constitutive activation of the LMP1 signaling pathway (14). The C-terminal cytoplasmic region was shown to interact with two families of proteins, TRAF (tumor necrosis factor [TNF] receptor-associated factor) and TRADD (TNF receptor-assoc...

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“…31 One of these two families of EBV microRNA is derived from BART, which were originally found to be expressed abundantly in NPC cells. 32 EBV expresses limited viral proteins in NPC cells, but encodes 22 BART pre-microRNAs which can be further What's new? Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated tumor in which only a few viral proteins but more than 20 BART microRNAs are detected, at abundant levels.…”

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confidence: 99%

Circulating Epstein–Barr virus microRNAs miR‐BART7 and miR‐BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment

Zhang

Zong

Shen

et al. 2014

Intl Journal of Cancer

102

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More than 75% of nasopharyngeal carcinoma (NPC) patients have already developed local or regional spread at diagnosis, which hampers effective treatment and results in a poor prognosis. It is essential to characterize more sensitive and specific biomarkers for screening of high risk individuals and assessment of NPC treatment effectiveness. NPC is an Epstein-Barr virus (EBV) associated tumor in which only a few viral proteins but more than 20 BamHI A rightward transcripts (BART) microRNAs are detected, at abundant levels. We hypothesized that these BART microRNAs may be novel biomarkers for NPC. Systematic analysis of EBV BART microRNA expression profiles in EBV latently infected Mutu I and Mutu III cell lines, EBV-harboring NPC and noncancerous NP cells found that miR-BART3, miR-BART7 and miR-BART13 microRNAs are highly expressed and regularly secreted into the extracellular environment of NPC cells. These BART microRNAs were evaluated for used as potential NPC biomarkers. Analysis of plasma specimens obtained from NPC patients (n 5 89), and healthy (n 5 28) and non-NPC tumor patient controls (n 5 18) found levels of both miR-BART7 and miR-BART13, but not miR-BART3, to be distinctly presence among NPC patients, with elevated levels being particularly apparent among patients with advanced disease. Receiver operating characteristic curve analysis combining miR-BART7 and miR-BART13 levels produces a 90% predictive value for the presence of NPC. Analysis of 41 NPC patients before and after radiotherapy showed that miR-BART7 and miR-BART13, but not miR-BART3, were diminished after treatment. These results indicate that EBV microRNAs, miR-BART7 and miR-BART13, may constitute useful new serological biomarkers for diagnosis of NPC and prediction of treatment efficacy.

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Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein

Cited by 21 publications

References 32 publications

Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene

Circulating Epstein–Barr virus microRNAs miR‐BART7 and miR‐BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment

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Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein

Cited by 21 publications

References 32 publications

Identification and prevalence of CD8+ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

Identification and prevalence of CD8+ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene

Circulating Epstein–Barr virus microRNAs miR‐BART7 and miR‐BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment

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Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2

Identification and prevalence of CD8⁺ T‐cell responses directed against Epstein‐Barr virus‐encoded latent membrane protein 1 and latent membrane protein 2