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1.
Nucleic Acids Res ; 47(6): 2807-2821, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30649516

RESUMO

Epstein-Barr virus proteins EBNA3A, EBNA3B and EBNA3C control hundreds of host genes after infection. Changes in epigenetic marks around EBNA3-regulated genes suggest that they exert transcriptional control in collaboration with epigenetic factors. The roles of polycomb repressive complex (PRC)2 subunit SUZ12 and of PRC1 subunit BMI1 were assessed for their importance in EBNA3-mediated repression and activation. ChIP-seq experiments for SUZ12 and BMI1 were performed to determine their global localization on chromatin and analysis offered further insight into polycomb protein distribution in differentiated cells. Their localization was compared to that of each EBNA3 to resolve longstanding questions about the EBNA3-polycomb relationship. SUZ12 did not co-localize with any EBNA3, whereas EBNA3C co-localized significantly and co-immunoprecipitated with BMI1. In cells expressing a conditional EBNA3C, BMI1 was sequestered to EBNA3C-binding sites after EBNA3C activation. When SUZ12 or BMI1 was knocked down in the same cells, SUZ12 did not contribute to EBNA3C-mediated regulation. Surprisingly, after BMI1 knockdown, EBNA3C repressed equally efficiently but host gene activation by EBNA3C was impaired. This overturns previous assumptions about BMI1/PRC1 functions during EBNA3C-mediated regulation, for the first time identifies directly a host factor involved in EBNA3-mediated activation and provides a new insight into how PRC1 can be involved in gene activation.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Interações Hospedeiro-Patógeno/genética , Complexo Repressor Polycomb 1/fisiologia , Ativação Transcricional , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Complexo Repressor Polycomb 1/metabolismo , Ligação Proteica
2.
PLoS Biol ; 15(8): e2001992, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28771465

RESUMO

Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that-after a phase of rapid proliferation-infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate <12 days post-infection and are unable to de-repress p18INK4c or BLIMP-1-in either newly infected cells or conditional LCLs-by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype. Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic persistence.


Assuntos
Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/fisiologia , Latência Viral , Linfócitos B/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Código das Histonas , Humanos , Imunoglobulinas/metabolismo , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Repressoras/metabolismo
3.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29367247

RESUMO

Epstein-Barr virus (EBV) establishes latent infection in human B cells and is associated with a wide range of cancers. The EBV nuclear antigen 3 (EBNA3) family proteins are critical for B cell transformation and function as transcriptional regulators. It is well established that EBNA3A and EBNA3C cooperate in the regulation of cellular genes. Here, we demonstrate that the gene STK39 is repressed only by EBNA3A. This is the first example of a gene regulated only by EBNA3A in EBV-transformed lymphoblastoid cell lines (LCLs) without the help of EBNA3C. This was demonstrated using a variety of LCLs carrying either knockout, revertant, or conditional EBNA3 recombinants. Investigating the kinetics of EBNA3A-mediated changes in STK39 expression showed that STK39 becomes derepressed quickly after EBNA3A inactivation. This derepression is reversible as EBNA3A reactivation represses STK39 in the same cells expressing a conditional EBNA3A. STK39 is silenced shortly after primary B cell infection by EBV, and no STK39-encoded protein (SPAK) is detected 3 weeks postinfection. Chromatin immunoprecipitation (ChIP) analysis indicates that EBNA3A directly binds to a regulatory region downstream of the STK39 transcription start site. For the first time, we demonstrated that the polycomb repressive complex 2 with the deposition of the repressive mark H3K27me3 is not only important for the maintenance of an EBNA3A target gene (STK39) but is also essential for the initial establishment of its silencing. Finally, we showed that DNA methyltransferases are involved in the EBNA3A-mediated repression of STK39IMPORTANCE EBV is well known for its ability to transform B lymphocytes to continuously proliferating lymphoblastoid cell lines. This is achieved in part by the reprogramming of cellular gene transcription by EBV transcription factors, including the EBNA3 proteins that play a crucial role in this process. In the present study, we found that EBNA3A epigenetically silences STK39 This is the first gene where EBNA3A has been found to exert its repressive role by itself, without needing its coregulators EBNA3B and EBNA3C. Furthermore, we demonstrated that the polycomb repressor complex is essential for EBNA3A-mediated repression of STK39 Findings in this study provide new insights into the regulation of cellular genes by the transcription factor EBNA3A.


Assuntos
Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Inativação Gênica , Herpesvirus Humano 4/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítio de Iniciação de Transcrição , Linfócitos B/patologia , Linfócitos B/virologia , Linhagem Celular Transformada , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Serina-Treonina Quinases/genética
4.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30135119

RESUMO

Epstein-Barr virus nuclear antigen 3C (EBNA3C) is a well-defined repressor of host gene expression in B cells transformed by Epstein-Barr virus (EBV) that cooperates with various cellular factors. It is established that EBNA3C interacts with the cellular factor RBPJ (RBP-Jκ or CBF1) through two distinct motifs: the TFGC motif, also called the homology domain (HD) motif, and the VWTP motif. In this study, we investigated the role of each motif in EBNA3C transcriptional repression activity by using two novel recombinant viruses with single RBPJ interaction motifs mutated (EBNA3C HDmut and EBNA3C W227S). Infection of primary B cells with either of these recombinant EBVs led to the successful establishment of lymphoblastoid cell lines (LCLs). Gene expression analysis showed that full repression of EBNA3C target genes is not achieved by EBNA3C HDmut compared to that with EBNA3C W227S or the EBNA3C wild type (WT). Focusing on the well-characterized EBNA3C-repressed genes COBLL1, ADAM28, and ADAMDEC1, we investigated the mechanism of EBNA3C-mediated transcriptional repression. Chromatin immunoprecipitation (ChIP) analysis indicated that EBNA3C HDmut is still able to recruit Polycomb proteins BMI1 and SUZ12 to COBLL1 as efficiently as EBNA3C WT does, leading to the full deposition of the repressive histone mark H3K27me3. However, we found that the activation-associated chromatin mark H3K4me3 is highly enriched at EBNA3C target genes in LCLs expressing EBNA3C HDmut. We show here that EBNA3C interacts with the histone lysine demethylase KDM2B and that this interaction is important for H3K4me3 removal and for the EBNA3C-mediated repression of COBLL1 and the ADAM28-ADAMDEC1 locus.IMPORTANCE EBV is a virus associated with human cancers and is well known for its ability to transform B lymphocytes into continuously proliferating lymphoblastoid cell lines. EBNA3C is considered an oncoprotein and has been shown to be essential for B cell transformation by EBV. EBNA3C is well characterized as a viral transcription factor, but very little is known about its mechanisms of action. In the present study, we demonstrate that removal of the activating histone mark H3K4me3 and deposition of the repressive mark H3K27me3 by EBNA3C on COBLL1 are achieved by at least two distinct mechanisms. Furthermore, we discovered that EBNA3C interacts with the lysine demethylase KDM2B and that this interaction is important for its transcriptional repressive function. The findings in this study provide new insights into the mechanism used by the oncoprotein EBNA3C to repress cellular target genes.


Assuntos
Proteínas ADAM/biossíntese , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Proteínas F-Box/metabolismo , Histonas/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição/biossíntese , Linfócitos B/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica/fisiologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteínas de Neoplasias , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo
5.
Nucleic Acids Res ; 45(5): 2368-2383, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-27903901

RESUMO

ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3-a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFß), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFß with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFß in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored.


Assuntos
Fatores de Ligação ao Core/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação da Expressão Gênica , Sítios de Ligação , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Subunidade beta de Fator de Ligação ao Core/metabolismo , Fatores de Ligação ao Core/fisiologia , Elementos Facilitadores Genéticos , Genoma Humano , Humanos , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
6.
PLoS Pathog ; 12(1): e1005383, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26751214

RESUMO

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression-particularly in relation to histone modifications and cell factors involved-the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.


Assuntos
Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Células Cultivadas , Cromatina/genética , Imunoprecipitação da Cromatina , Interações Hospedeiro-Parasita/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
PLoS Pathog ; 11(7): e1005031, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26153983

RESUMO

We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours--including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.


Assuntos
Transformação Celular Neoplásica/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , MicroRNAs/genética , Linfócitos B/virologia , Western Blotting , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p57/biossíntese , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imunoprecipitação , MicroRNAs/biossíntese , Oncogenes , Reação em Cadeia da Polimerase em Tempo Real
8.
J Virol ; 89(10): 5222-37, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25787276

RESUMO

UNLABELLED: Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Variação Genética , Genoma Viral , Herpesvirus Humano 4/genética , Sequência de Aminoácidos , Antígenos Virais/genética , Portador Sadio/virologia , Linhagem Celular Tumoral , DNA Viral/genética , Epitopos de Linfócito T/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/classificação , Herpesvirus Humano 4/isolamento & purificação , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Proteínas da Matriz Viral/genética
9.
Curr Top Microbiol Immunol ; 391: 61-117, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26428372

RESUMO

Epstein-Barr virus nuclear antigens EBNA3A , EBNA3B and EBNA3C are a family of three large latency-associated proteins expressed in B cells induced to proliferate by the virus. Together with the other nuclear antigens (EBNA-LP, EBNA2 and EBNA1), they are expressed from a polycistronic transcription unit that is probably unique to B cells. However, compared with the other EBNAs, hitherto the EBNA3 proteins were relatively neglected and their roles in EBV biology rather poorly understood. In recent years, powerful new technologies have been used to show that these proteins are central to the latency of EBV in B cells, playing major roles in reprogramming the expression of host genes affecting cell proliferation, survival, differentiation and immune surveillance. This indicates that the EBNA3s are critical in EBV persistence in the B cell system and in modulating B cell lymphomagenesis. EBNA3A and EBNA3C are necessary for the efficient proliferation of EBV-infected B cells because they target important tumour suppressor pathways--so operationally they are considered oncoproteins. In contrast, it is emerging that EBNA3B restrains the oncogenic capacity of EBV, so it can be considered a tumour suppressor--to our knowledge the first to be described in a tumour virus. Here, we provide a general overview of the EBNA3 genes and proteins. In particular, we describe recent research that has highlighted the complexity of their functional interactions with each other, with specific sites on the human genome and with the molecular machinery that controls transcription and epigenetic states of diverse host genes.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Família Multigênica , Proteínas Oncogênicas Virais/genética , Proteínas Supressoras de Tumor/genética
10.
PLoS Pathog ; 9(2): e1003187, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23436997

RESUMO

To explore the role of p16(INK4a) as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a) and p14(ARF). Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16(INK4a) protein but expressing a functional 14(ARF)-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional p16(INK4a). Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a) and growth arrest, EBNA3C inactivation in the p16(INK4a)-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a)-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a) expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a)-null, functional EBNA3C is dispensable for the outgrowth of LCLs.


Assuntos
Linfócitos B/virologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Repressão Epigenética/genética , Herpesvirus Humano 4/fisiologia , Ativação Linfocitária , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Loci Gênicos , Herpesvirus Humano 4/imunologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Cultura Primária de Células , Latência Viral
11.
Nucleic Acids Res ; 40(15): 7233-46, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22584624

RESUMO

Detailed analyses of the chromatin around the BIM promoter has revealed that latent Epstein-Barr virus (EBV) triggers the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of histone H3 lysine 27 (H3K27me3) at this locus. The recruitment is absolutely dependent on nuclear proteins EBNA3A and EBNA3C; what is more, epitope-tagged EBNA3C could be shown bound near the transcription start site (TSS). EBV induces no consistent changes in the steady-state expression of PRC2 components, but lentivirus delivery of shRNAs against PRC2 and PRC1 subunits disrupted EBV repression of BIM. The activation mark H3K4me3 is largely unaltered at this locus irrespective of H3K27me3 status, suggesting the establishment of a 'bivalent' chromatin domain. Consistent with the 'poised' nature of these domains, RNA polymerase II (Pol II) occupancy was not altered by EBV at the BIM TSS, but analysis of phospho-serine 5 on Pol II indicated that EBNA3A and EBNA3C together inhibit initiation of BIM transcripts. B cell lines carrying EBV encoding a conditional EBNA3C-oestrogen receptor-fusion revealed that this epigenetic repression of BIM was reversible, but took more than 3 weeks from when EBNA3C was inactivated.


Assuntos
Antígenos Virais/metabolismo , Proteínas Reguladoras de Apoptose/genética , Herpesvirus Humano 4/fisiologia , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas do Grupo Polycomb , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Transcrição Gênica
12.
J Virol ; 86(3): 1683-95, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22090140

RESUMO

Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq-c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38. This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.


Assuntos
Epigênese Genética , Linfoma/virologia , Mardivirus/genética , Linfócitos T/virologia , Latência Viral/genética , Animais , Sequência de Bases , Aves , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA , Primers do DNA , Humanos , Linfoma/patologia , Mardivirus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
PLoS Pathog ; 6(6): e1000951, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20548956

RESUMO

As an inhibitor of cyclin-dependent kinases, p16(INK4A) is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16(INK4A) to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16(INK4A) transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16(INK4A) and have a similar pattern of histone modifications on p16(INK4A) as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16(INK4A) requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16(INK4A) by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16(INK4A) DNA methylation during B cell lymphomagenesis.


Assuntos
Oxirredutases do Álcool/metabolismo , Antígenos Virais/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Oxirredutases do Álcool/genética , Antígenos Virais/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Proliferação de Células , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional
14.
PLoS Pathog ; 5(6): e1000492, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19557159

RESUMO

In human B cells infected with Epstein-Barr virus (EBV), latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP) and bisulphite sequencing of regions within the large CpG island located at the 5' end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL) biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27) and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Linfócitos B/fisiologia , Linfócitos B/virologia , Metilação de DNA , Herpesvirus Humano 4/fisiologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Latência Viral/fisiologia , Acetilação , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos B/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Expressão Gênica , Genes Supressores de Tumor , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Estabilidade de RNA , Análise de Sequência de DNA , Células Tumorais Cultivadas
15.
Semin Cancer Biol ; 19(6): 366-76, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19635566

RESUMO

A defining characteristic of the aggressive B cell tumour Burkitt's lymphoma (BL) is a reciprocal chromosomal translocation that activates the Myc oncogene by juxtaposing it to one of the immunoglobulin gene loci. The consequences of activating Myc include cell growth and proliferation that can lead to lymphomagenesis; however, as part of a fail-safe mechanism that has evolved in metazoans to reduce the likelihood of neoplastic disease, activated oncogenes such as Myc may also induce cell death by apoptosis and/or an irreversible block to proliferation called senescence. For lymphoma to develop it is necessary that these latter processes are repressed. More than 95% of a subset of BL - known as endemic (e)BL because they are largely restricted to regions of equatorial Africa and similar geographical regions - carry latent Epstein-Barr virus (EBV) in the form of nuclear extra-chromosomal episomes. Although EBV is not generally regarded as a driving force of BL cell proliferation, it plays an important role in the pathogenesis of eBL. Latency-associated EBV gene products can inhibit a variety of pathways that lead to apoptosis and senescence; therefore EBV probably counteracts the proliferation-restricting activities of deregulated Myc and so facilitates the development of BL.


Assuntos
Linfoma de Burkitt/virologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/patogenicidade , Proteínas Proto-Oncogênicas c-myc/metabolismo , Latência Viral/fisiologia , Animais , Humanos , Camundongos
16.
J Virol ; 83(21): 11142-51, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19692466

RESUMO

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B Meq(BZIP/BZIP)) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B Meq(Hom/Hom)). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.


Assuntos
Transformação Celular Viral , Linfoma de Células T/virologia , Mardivirus , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Animais , Galinhas/virologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Zíper de Leucina , Mardivirus/química , Mardivirus/metabolismo , Mardivirus/patogenicidade , Doença de Marek/virologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas Virais/genética , Doenças das Aves Domésticas/virologia , Multimerização Proteica , Reprodutibilidade dos Testes , Alinhamento de Sequência , Análise de Sobrevida , Latência Viral
17.
Elife ; 62017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28425914

RESUMO

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis.


Assuntos
Apoptose , Linfócitos B/fisiologia , Linfócitos B/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Humanos , Camundongos
18.
Cell Host Microbe ; 22(1): 61-73.e7, 2017 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-28704654

RESUMO

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication.


Assuntos
Coinfecção , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/patogenicidade , Neoplasias/virologia , Animais , Linfócitos B/virologia , Linhagem Celular Tumoral , Citocinas/sangue , DNA Viral/análise , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Genes Virais/genética , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma de Efusão Primária/etiologia , Linfoma de Efusão Primária/virologia , Camundongos , Baço/patologia , Baço/virologia , Taxa de Sobrevida , Replicação Viral
19.
J Exp Med ; 213(6): 921-8, 2016 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-27217538

RESUMO

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.


Assuntos
Linfoma de Burkitt/imunologia , Citidina Desaminase/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B/imunologia , Herpesvirus Humano 4/imunologia , Proteínas de Neoplasias/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Linfoma de Burkitt/genética , Linhagem Celular , Citidina Desaminase/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Rearranjo Gênico do Linfócito B/genética , Herpesvirus Humano 4/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Elementos de Resposta/imunologia , Hipermutação Somática de Imunoglobulina/genética
20.
Oncogene ; 22(46): 7181-91, 2003 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-14562046

RESUMO

Epstein-Barr virus (EBV) is involved in the pathogenesis of several B cell lymphoproliferations, but the precise contribution it makes to the aetiology of each remains unclear. In vitro, the virus has potent growth transforming activity and efficiently induces the continuous proliferation of normal human B cells. A comparison of EBV-infected primary B cells with an isogenic population induced to proliferate by CD40-ligand (CD40L) and IL4 has revealed that EBV can override - by a novel mechanism - the p53/pRb-mediated G1 checkpoint activated in normal B cells by a genotoxic stress. In cells responding to cisplatin, although p53 is stabilized and activated, EBV latent gene expression appears to inhibit the accumulation of newly synthesized p21(WAF1/CIP1) and the downregulation of cyclin D2 that occur in the normal cells. Consequently, in the EBV-infected cells, CDK2 remains active, hyperphosphorylation of pRb is maintained and the replication of damaged DNA can occur. Under conditions of severe genomic stress, this absence of p21(WAF1/CIP1) function can result in apoptosis; however, when damage is less sustained, genomic instability may arise and this in turn could contribute to the development of a variety of EBV-associated B cell malignancies.


Assuntos
Linfócitos B/imunologia , Ligante de CD40/farmacologia , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclo Celular/imunologia , Fase G1/efeitos dos fármacos , Genes p53/efeitos dos fármacos , Herpesvirus Humano 4/fisiologia , Mutagênicos/toxicidade , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Bromodesoxiuridina , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quinase 2 Dependente de Ciclina , Citometria de Fluxo , Fase G1/fisiologia , Humanos , Interleucina-4/farmacologia , Modelos Biológicos , Ribonucleases/metabolismo
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