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Nuclear factor kappa B-dependent activation of the antiapoptotic bfl-1 gene by the Epstein-Barr virus latent membrane protein 1 and activated CD40 receptor.

ABSTRACT: Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000). We now report that LMP1 drives bfl-1 promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-kappa B dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increased bfl-1 mRNA levels and an NF-kappa B-dependent increase in bfl-1 promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-kappa B-like binding motif that is essential for transactivation of bfl-1 by LMP1, CD40, and the NF-kappa B subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties.

SUBMITTER: D'Souza BN

PROVIDER: S-EPMC369510 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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Nuclear factor kappa B-dependent activation of the antiapoptotic bfl-1 gene by the Epstein-Barr virus latent membrane protein 1 and activated CD40 receptor.

D'Souza Brendan N BN Edelstein Leonard C LC Pegman Pamela M PM Smith Sinéad M SM Loughran Sinéad T ST Clarke Ann A Mehl Anja A Rowe Martin M Gélinas Céline C Walls Dermot D

Journal of virology 20040201 4

Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibitin ...[more]

PMID: 14747545

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Project description:The Epstein Barr virus (EBV) encoded latent membrane protein-1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of Nuclear Factor-kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis, and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative RT-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an OCT-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response. Keywords: Analysis of gene expression changes altered by the microRNA, miR-146a EBV positive Burkitt's lymphoma cell line, Akata, was infected with the control retrovirus, pEhyg, or the miR-146a expressing retrovirus, pEhyg-miR-146a. Two infections were carried out for control retrovirus and two infections were carried out with the pEhyg-miR-146a retrovirus. Total RNA was prepared from each of the 4 infections. Control and miR-146a infection set 1 was subjected to dual color array analysis on chip 1. Control and miR-146a infection set 2 was subjected to dual color array analysis on second array of chip 1. Dye swaps of each of these were carried out on two arrays from a second chip (chip 2).

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.

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Project description:Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers.IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology.

Dataset Information

Nuclear factor kappa B-dependent activation of the antiapoptotic bfl-1 gene by the Epstein-Barr virus latent membrane protein 1 and activated CD40 receptor.

Publications

Nuclear factor kappa B-dependent activation of the antiapoptotic bfl-1 gene by the Epstein-Barr virus latent membrane protein 1 and activated CD40 receptor.

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