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Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation.

ABSTRACT: The Epstein-Barr virus (EBV), a human B-lymphotropic gamma herpesvirus, contains multiple repetitive sequences within its genome. A group of repetitive sequences, known as the family of repeats (FR), contains multiple binding sites for the viral trans-acting protein EBNA-1. The FR sequences are important for viral genome maintenance and for the regulation of the promoter involved in viral latent gene expression. It has been reported that a palindromic sequence with a putative secondary structure exists at the 3' end of the FR in the genome of the EBV B95-8 strain and that this palindromic sequence has been deleted from the FR of the commonly used EBV miniplasmids. For the first time, we cloned an EBV B95-8 DNA fragment containing the full-length FR, which enabled us to examine the functional difference between full-length and deleted FRs. The full-length FR, like the deleted FR, functioned as a transcriptional enhancer of the viral latent gene promoter, but that transactivation was significantly attenuated in the case of the full-length FR. No significant enhancement of replication was observed when the deleted FR was replaced with the full-length FR in an EBV miniplasmid. By contrast, when the same set of FR sequences were tested in the context of the complete EBV genome, the full-length FR resulted in more-efficient B-cell transformation than the deleted FR. We propose that the presence of the full-length FR contributes to the precise regulation of the viral latent promoter and increases the efficiency of B-cell transformation.

SUBMITTER: Ali AK

PROVIDER: S-EPMC2738248 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation.

Ali Ahmed K M AK Saito Satoru S Shibata Sachiko S Takada Kenzo K Kanda Teru T

Journal of virology 20090701 18

The Epstein-Barr virus (EBV), a human B-lymphotropic gamma herpesvirus, contains multiple repetitive sequences within its genome. A group of repetitive sequences, known as the family of repeats (FR), contains multiple binding sites for the viral trans-acting protein EBNA-1. The FR sequences are important for viral genome maintenance and for the regulation of the promoter involved in viral latent gene expression. It has been reported that a palindromic sequence with a putative secondary structure ...[more]

PMID: 19570868

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Project description:BackgroundThe importance of the malignant cell environment to its growth and survival is becoming increasingly apparent, with dynamic cross talk between the neoplastic cell, the leukocyte infiltrate and the stroma. Most cancers are accompanied by leukocyte infiltration which, contrary to an anticipated immuno-protective role, could be contributing to tumour development and cancer progression. Epstein-Barr virus (EBV) associated cancers, including nasopharyngeal carcinoma and Hodgkin's Disease, show a considerable leukocyte infiltration which surrounds the neoplastic cells, raising the questions as to what role these cells play in either restricting or supporting the tumour and what draws the cells into the tumour. In order to begin to address this we have studied a transgenic model of multistage carcinogenesis with epithelial expression of the EBV primary oncoprotein, latent membrane protein 1 (LMP1). LMP1 is expressed particularly in the skin, which develops a hyperplastic pathology soon after birth.ResultsThe pathology advances with time leading to erosive dermatitis which is inflamed with a mixed infiltrate involving activated CD8+ T-cells, CD4+ T-cells including CD4+/CD25+/FoxP3+ Treg cells, mast cells and neutrophils. Also significant dermal deposition of immunoglobulin-G (IgG) is observed as the pathology advances. Along with NF-kappaB activation, STAT3, a central factor in inflammation regulation, is activated in the transgenic tissue. Several inflammatory factors are subsequently upregulated, notably CD30 and its ligand CD153, also leukocyte trafficking factors including CXCL10, CXCL13, L-selectin and TGF?1, and inflammatory cytokines including IL-1?, IL-3 and the murine IL-8 analogues CXCL1, CXCL2 and CXCL5-6, amongst others. The crucial role of mature T- and/or B-lymphocytes in the advancing pathology is demonstrated by their elimination, which precludes mast cell infiltration and limits the pathology to an early, benign stage.ConclusionsLMP1 can lead to the activation of several key factors mediating proliferation, angiogenesis and inflammation in vivo. With the initiation of an inflammatory programme, leukocyte recruitment follows which then itself contributes to the progressing pathology in these transgenic mice, with a pivotal role for B-and/or T-cells in the process. The model suggests a basis for the leukocyte infiltrate observed in EBV-associated cancer and its supporting role, as well as potential points for therapeutic intervention.

Project description:Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkin's and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitt's lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1- and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids, and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could be used in the treatment of LMP1+ EBV-associated malignancies by targeting an LMP1-specific dependency on lipogenesis.IMPORTANCE Despite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational, and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of virus-associated malignancies. Noncancerous cells preferentially obtain fatty acids from dietary sources, whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis is an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

Project description:In this study, we demonstrate that expression of viral latent membrane protein 1 (LMP1) in a mouse B cell line renders the animals responsive to protection from a 38C13-LMP1 tumor challenge with a novel vaccine. The Epstein-Barr virus (EBV) preferentially infects circulating B lymphocytes, has oncogenic potential, and is associated with a wide variety of B cell lymphomas. EBV is ectotrophic to human cells, and currently there are no B cell animal models of EBV-associated lymphoma that can be used to investigate vaccine immunotherapy. Since most EBV-infected human tumor cells express latent membrane protein 1 (LMP1) on their surface, this viral antigen was tested as a potential target for an anticancer vaccine in a mouse model. Here, we describe a new mouse model of LMP1-expressing B cell lymphoma produced with plasmid transduction of 38C13 into mouse B cells. The expression of LMP-1 was confirmed with a western blot analysis and immunocytochemistry. We then designed a novel LMP1 vaccine, by fusing viral antigen LMP1 surface loop epitopes to the surface of a viral antigen carrier, the Tobacco Mosaic virus (TMV). Vaccinated mice produced high titer antibodies against the TMV-LMP1 vaccine; however, cellular responses were at the baseline, as measured with IFNγ ELISpot. Despite this, the vaccine showed significant protection from a 38C13-LMP1 tumor challenge. To provide additional immune targets, we compared TMV-LMP1 peptide immunization with DNA immunization with the full-length LMP1 gene. Anti-LMP1 antibodies were significantly higher in TMV-LMP1-vaccinated mice compared to the DNA-immunized mice, but, as predicted, DNA-vaccinated mice had improved cellular responses using IFNγ ELISpot. Surprisingly, the TMV-LMP1 vaccine provided protection from a 38C13-LMP1 tumor challenge, while the DNA vaccine did not. Thus, we demonstrated that LMP1 expression in a mouse B cell line is responsive to antibody immunotherapy that may be applied to EBV-associated disease.

Project description:To examine the risk of late-onset post-transplant lymphoproliferative disorder (PTLD) in the presence of persisting high Epstein-Barr virus (EBV) in EBV naïve pediatric heart transplant (HT) recipients.A retrospective review of the medical records of the 145 pediatric HT recipients who had serial EBV viral load monitoring at our center was performed. We defined EBV naive patients whose EBV serology either IgM or IgG in the blood were negative at the time of HT and excluded passive transmission from mother to child in subjects less than 6 mo of age.PTLD was diagnosed in 8 out of 145 patients (5.5%); 6/91 (6.5%) in those who were EBV seropositive and 2/54 (3.7%) in the EBV naïve group at the time of HT (P = 0.71). We found 32/145 (22%) patients with persistently high EBV load during continuing follow-up; 20/91 (22%) in EBV seropositive group vs 12/54 (22%) in EBV naïve group (P = 0.97). There was no significant association between pre-HT serostatus and EBV load after transplant (P > 0.05). In the EBV seropositive group, PTLD was diagnosed in 15% (3/20) of patients with high EBV vs 4.2% (3/71) of patients with low or undetectable EBV load (P = 0.14) whereas in EBV naïve patients 8.3% (1/12) of those with high EBV load and 2.3% (1/42) with low or undetectable EBV load (P = 0.41). There was a highly significant association between occurrence of PTLD in those with high EBV load and duration of follow up (4.3 ± 3.9 years) after HT by Cochran-Armitage test for the entire cohort (P = 0.005). At least one episode of acute rejection occurred in 72% (23/32) of patients with high EBV vs 36% (41/113) patients with low or undetectable EBV after HT (P < 0.05).There is an association between persistently high EBV load during post-HT follow up and the occurrence of late-onset PTLD in pediatric HT recipients irrespective of serostatus at the time of transplant. The occurrence of allograft rejection increased in patients with high EBV load presumably due to reduction in immunosuppression.

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Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation.

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Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation.

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