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The cellular ataxia telangiectasia-mutated kinase promotes epstein-barr virus lytic reactivation in response to multiple different types of lytic reactivation-inducing stimuli.

ABSTRACT: The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor ? (TGF-?) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.

SUBMITTER: Hagemeier SR

PROVIDER: S-EPMC3503132 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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The cellular ataxia telangiectasia-mutated kinase promotes epstein-barr virus lytic reactivation in response to multiple different types of lytic reactivation-inducing stimuli.

Hagemeier Stacy R SR Barlow Elizabeth A EA Meng Qiao Q Kenney Shannon C SC

Journal of virology 20120926 24

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular D ...[more]

PMID: 23015717

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Project description:Epstein-Barr virus (EBV) infects 95% of adults worldwide and causes infectious mononucleosis. EBV is associated with endemic Burkitt lymphoma, Hodgkin lymphoma, posttransplant lymphomas, nasopharyngeal and gastric carcinomas. In these cancers and in most infected B-cells, EBV maintains a state of latency, where nearly 80 lytic cycle antigens are epigenetically suppressed. To gain insights into host epigenetic factors necessary for EBV latency, we recently performed a human genome-wide CRISPR screen that identified the chromatin assembly factor CAF1 as a putative Burkitt latency maintenance factor. CAF1 loads histones H3 and H4 onto newly synthesized host DNA, though its roles in EBV genome chromatin assembly are uncharacterized. Here, we found that CAF1 depletion triggered lytic reactivation and virion secretion from Burkitt cells, despite also strongly inducing interferon-stimulated genes. CAF1 perturbation diminished occupancy of histones 3.1 and 3.3 and of repressive histone 3 lysine 9 and 27 trimethyl (H3K9me3 and H3K27me3) marks at multiple viral genome lytic cycle regulatory elements. Suggestive of an early role in establishment of latency, EBV strongly upregulated CAF1 expression in newly infected primary human B-cells prior to the first mitosis, and histone 3.1 and 3.3 were loaded on the EBV genome by this time point. Knockout of CAF1 subunit CHAF1B impaired establishment of latency in newly EBV-infected Burkitt cells. A nonredundant latency maintenance role was also identified for the DNA synthesis-independent histone 3.3 loader histone regulatory homologue A (HIRA). Since EBV latency also requires histone chaperones alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX) and death domain-associated protein (DAXX), EBV coopts multiple host histone pathways to maintain latency, and these are potential targets for lytic induction therapeutic approaches.IMPORTANCE Epstein-Barr virus (EBV) was discovered as the first human tumor virus in endemic Burkitt lymphoma, the most common childhood cancer in sub-Saharan Africa. In Burkitt lymphoma and in 200,000 EBV-associated cancers per year, epigenetic mechanisms maintain viral latency, during which lytic cycle factors are silenced. This property complicated EBV's discovery and facilitates tumor immunoevasion. DNA methylation and chromatin-based mechanisms contribute to lytic gene silencing. Here, we identified histone chaperones CAF1 and HIRA, which have key roles in host DNA replication-dependent and replication-independent pathways, respectively, as important for EBV latency. EBV strongly upregulates CAF1 in newly infected B-cells, where viral genomes acquire histone 3.1 and 3.3 variants prior to the first mitosis. Since histone chaperones ATRX and DAXX also function in maintenance of EBV latency, our results suggest that EBV coopts multiple histone pathways to reprogram viral genomes and highlight targets for lytic induction therapeutic strategies.

Project description:Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

Dataset Information

The cellular ataxia telangiectasia-mutated kinase promotes epstein-barr virus lytic reactivation in response to multiple different types of lytic reactivation-inducing stimuli.

Publications

The cellular ataxia telangiectasia-mutated kinase promotes epstein-barr virus lytic reactivation in response to multiple different types of lytic reactivation-inducing stimuli.

Similar Datasets

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