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1.
Cell ; 184(3): 643-654.e13, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33482082

RESUMO

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.


Assuntos
Ciclo Celular , Reagentes de Ligações Cruzadas/química , DNA Viral/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Plasmídeos/metabolismo , Origem de Replicação , Replicação Viral/fisiologia , Sequência de Aminoácidos , Linfócitos B/metabolismo , Linhagem Celular , Adutos de DNA/metabolismo , Replicação do DNA , Endonucleases/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Humanos , Mutação/genética , Ligação Proteica , Recombinação Genética/genética , Tirosina/metabolismo
2.
PLoS Pathog ; 19(6): e1010478, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37262099

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.


Assuntos
Infecções por Vírus Epstein-Barr , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Lisina/genética , Proteômica , Replicação do DNA , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Replicação Viral , Estabilidade Proteica , Plasmídeos , Origem de Replicação
3.
PLoS Pathog ; 17(1): e1009231, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33471863

RESUMO

Liquid-liquid phase separation (LLPS) can drive formation of diverse and essential macromolecular structures, including those specified by viruses. Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genomes associate with the viral encoded Latency-Associated Nuclear Antigen (LANA) to form stable nuclear bodies (NBs) during latent infection. Here, we show that LANA-NB formation and KSHV genome conformation involves LLPS. Using LLPS disrupting solvents, we show that LANA-NBs are partially disrupted, while DAXX and PML foci are highly resistant. LLPS disruption altered the LANA-dependent KSHV chromosome conformation but did not stimulate lytic reactivation. We found that LANA-NBs undergo major morphological transformation during KSHV lytic reactivation to form LANA-associated replication compartments encompassing KSHV DNA. DAXX colocalizes with the LANA-NBs during latency but is evicted from the LANA-associated lytic replication compartments. These findings indicate the LANA-NBs are dynamic super-molecular nuclear structures that partly depend on LLPS and undergo morphological transitions corresponding to the different modes of viral replication.


Assuntos
Antígenos Virais/química , Proteínas Correpressoras/metabolismo , Genoma Viral/genética , Herpesvirus Humano 8/genética , Corpos de Inclusão Intranuclear/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/química , Sarcoma de Kaposi/virologia , Antígenos Virais/genética , Linhagem Celular Tumoral , Herpesvirus Humano 8/fisiologia , Histonas/metabolismo , Humanos , Corpos de Inclusão Viral/química , Corpos de Inclusão Viral/metabolismo , Corpos de Inclusão Intranuclear/química , Infecção Latente , Extração Líquido-Líquido , Proteínas Nucleares/genética , Plasmídeos/genética , Latência Viral , Replicação Viral
4.
PLoS Pathog ; 17(1): e1009208, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33497421

RESUMO

Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers.


Assuntos
Linfócitos B/patologia , Transformação Celular Viral , Cromatina/genética , Infecções por Vírus Epstein-Barr/patologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Nucleotídeos/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Cromatina/metabolismo , Epigênese Genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Metaboloma , Transcriptoma , Proteínas Virais/genética
5.
EMBO J ; 35(2): 193-207, 2016 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-26658110

RESUMO

Telomeres and tumor suppressor protein TP53 (p53) function in genome protection, but a direct role of p53 at telomeres has not yet been described. Here, we have identified non-canonical p53-binding sites within the human subtelomeres that suppress the accumulation of DNA damage at telomeric repeat DNA. These non-canonical subtelomeric p53-binding sites conferred transcription enhancer-like functions that include an increase in local histone H3K9 and H3K27 acetylation and stimulation of subtelomeric transcripts, including telomere repeat-containing RNA (TERRA). p53 suppressed formation of telomere-associated γH2AX and prevented telomere DNA degradation in response to DNA damage stress. Our findings indicate that p53 provides a direct chromatin-associated protection to human telomeres, as well as other fragile genomic sites. We propose that p53-associated chromatin modifications enhance local DNA repair or protection to provide a previously unrecognized tumor suppressor function of p53.


Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA/genética , Telômero/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Transporte/genética , Células HCT116 , Humanos , Ligação Proteica , Telômero/genética , Proteína Supressora de Tumor p53/genética
6.
J Virol ; 93(20)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31142669

RESUMO

Epstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised oriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to the oriP Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the oriP Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCE Epstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element in oriP is the essential minimal replicator of oriP Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.


Assuntos
Replicação do DNA , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Origem de Replicação , Replicação Viral , Sequência de Bases , Sítios de Ligação , Antígenos Nucleares do Vírus Epstein-Barr/genética , Humanos , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
7.
J Virol ; 91(20)2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28794029

RESUMO

Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state.IMPORTANCE Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.

8.
J Virol ; 90(11): 5353-5367, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27009953

RESUMO

UNLABELLED: Epstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation. IMPORTANCE: EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Fator C1 de Célula Hospedeira/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Plasmídeos , Latência Viral/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Histonas/genética , Histonas/metabolismo , Fator C1 de Célula Hospedeira/deficiência , Fator C1 de Célula Hospedeira/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Humanos , Metilação , Fator 2 de Transcrição de Octâmero/genética , Origem de Replicação
9.
Mol Cell ; 35(4): 403-13, 2009 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-19716786

RESUMO

Telomere-repeat-encoding RNA (referred to as TERRA) has been identified as a potential component of yeast and mammalian telomeres. We show here that TERRA RNA interacts with several telomere-associated proteins, including telomere repeat factors 1 (TRF1) and 2 (TRF2), subunits of the origin recognition complex (ORC), heterochromatin protein 1 (HP1), histone H3 trimethyl K9 (H3 K9me3), and members of the DNA-damage-sensing pathway. siRNA depletion of TERRA caused an increase in telomere dysfunction-induced foci, aberrations in metaphase telomeres, and a loss of histone H3 K9me3 and ORC at telomere repeat DNA. Previous studies found that TRF2 amino-terminal GAR domain recruited ORC to telomeres. We now show that TERRA RNA can interact directly with the TRF2 GAR and ORC1 to form a stable ternary complex. We conclude that TERRA facilitates TRF2 interaction with ORC and plays a central role in telomere structural maintenance and heterochromatin formation.


Assuntos
Heterocromatina/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , RNA Nuclear/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Sítios de Ligação , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Aberrações Cromossômicas , Metilação de DNA , Células HCT116 , Histonas/metabolismo , Humanos , Estrutura Terciária de Proteína , Interferência de RNA , Telômero/ultraestrutura , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Fatores de Tempo , Transfecção
10.
PLoS Pathog ; 7(8): e1002140, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21876668

RESUMO

Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5' and 3' end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Genes Virais/fisiologia , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , Proteínas Repressoras/fisiologia , Transativadores/genética , Latência Viral/fisiologia , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Genes Reguladores/genética , Herpesvirus Humano 8/imunologia , Humanos , Linfoma de Células B/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Dedos de Zinco/fisiologia , Coesinas
11.
PLoS Pathog ; 6(8): e1001048, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20730088

RESUMO

The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound approximately 40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection.


Assuntos
Epigênese Genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/genética , Regiões Promotoras Genéticas/genética , Latência Viral/genética , Fator de Ligação a CCCTC , Linhagem Celular , Cromatina/genética , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Infecções por Vírus Epstein-Barr/genética , Perfilação da Expressão Gênica , Genes Virais , Humanos , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
12.
J Biol Chem ; 285(43): 33358-33370, 2010 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-20716523

RESUMO

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3'-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.


Assuntos
Herpesvirus Humano 4/fisiologia , MicroRNAs/metabolismo , Edição de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Latência Viral/fisiologia , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Inativação Gênica/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , MicroRNAs/genética , RNA Viral/genética , Ribonuclease III/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
J Virol ; 82(9): 4647-55, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18305033

RESUMO

Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/química , Herpesvirus Humano 4/fisiologia , Transativadores/fisiologia , Proteínas Virais/fisiologia , Replicação Viral , Linhagem Celular , DNA Mitocondrial/biossíntese , Humanos , Proteínas Imediatamente Precoces , Proteínas Mitocondriais , Proteínas Nucleares , Ligação Proteica
14.
Oncotarget ; 10(38): 3581-3591, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31217894

RESUMO

Subtelomeric transcription and chromatin can have a significant impact on telomere repeat maintenance and chromosome stability. We have previously found that tumor suppressor protein p53 (TP53) can bind to retrotransposon-like elements in a majority of human subtelomeres to regulate TERRA transcription and telomeric histone acetylation in response to DNA damage. TP53 also prevents the accumulation of γH2AX DNA-damage signaling at telomeres. We now show that the inherited TP53 polymorphism Pro47Ser (hereafter S47), which is enriched in populations of African descent, is associated with elevated marks of telomere dysfunction. We found that human and mouse cells carrying the S47 variant show increased γH2AX DNA-damage signals at telomeres, as well as reduced TERRA transcription and subtelomeric histone acetylation in response to DNA damage stress. Cell-lines containing inducible genes for P47 or S47 versions of p53, as well mouse embryo fibroblasts (MEFs) reconstituted with human p53, showed elevated telomere-induced DNA damage foci and metaphase telomere signal loss in cells with S47. Human lymphoblastoid cell lines (LCLs) derived from individuals homozygous for S47, show increased accumulation of subtelomeric γH2AX and unstable telomere repeats in response to DNA damage relative to age matched LCLs homozygous for P47. Furthermore, LCLs with S47 had reduced replicative lifespan. These studies indicate that the naturally occurring S47 variant of p53 can affect telomeric chromatin, telomere repeat stability, and replicative capacity. We discuss the potential evolutionary significance of the S47 variant to African populations with respect to telomere regulation and the implications for inherited health disparities.

15.
Oncotarget ; 10(43): 4505-4506, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31327987

RESUMO

[This corrects the article DOI: 10.18632/oncotarget.14540.].

16.
Oncotarget ; 8(5): 7248-7264, 2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-28077791

RESUMO

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.


Assuntos
Carcinoma/virologia , Transformação Celular Viral , DNA Viral/genética , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virologia , Latência Viral , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Carcinoma/metabolismo , Carcinoma/patologia , Cristalografia por Raios X , Replicação do DNA , DNA Viral/biossíntese , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Células HeLa , Herpesvirus Humano 4/crescimento & desenvolvimento , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Plasmídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Survivina , Replicação Viral
17.
Nat Commun ; 7: 12707, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27581705

RESUMO

The histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. Here we report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through non-conserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host-antiviral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an antiviral histone chaperone to promote viral latency and cellular immortalization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Herpesvirus Humano 4/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas do Envelope Viral/metabolismo , Linfócitos B/imunologia , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Correpressoras , Células HEK293 , Humanos , Chaperonas Moleculares , Ligação Proteica/genética , Domínios Proteicos , Latência Viral/genética
18.
PLoS One ; 6(5): e19596, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21573113

RESUMO

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinases , Linhagem Celular Tumoral , Centrômero/metabolismo , Aberrações Cromossômicas , DNA de Neoplasias/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Fase G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Metáfase , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Proteômica , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-Like
19.
PLoS One ; 2(12): e1257, 2007 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18040525

RESUMO

Recombination-like structures formed at origins of DNA replication may contribute to replication fidelity, sister chromatid cohesion, chromosome segregation, and overall genome stability. The Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) provides episomal genome stability through a poorly understood mechanism. We show here that recombinational repair proteins MRE11 and NBS1 are recruited to the Dyad Symmetry (DS) region of OriP in a TRF2- and cell cycle-dependent manner. Depletion of MRE11 or NBS1 by siRNA inhibits OriP replication and destabilized viral episomes. OriP plasmid maintenance was defective in MRE11 and NBS1 hypomorphic fibroblast cell lines and only integrated, non-episomal forms of EBV were detected in a lympoblastoid cell line derived from an NBS1-mutated individual. Two-dimensional agarose gel analysis of OriP DNA revealed that recombination-like structures resembling Holliday-junctions form at OriP in mid S phase. MRE11 and NBS1 association with DS coincided with replication fork pausing and origin activation, which preceded the formation of recombination structures. We propose that NBS1 and MRE11 promote replication-associated recombination junctions essential for EBV episomal maintenance and genome stability.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Herpesvirus Humano 4/fisiologia , Proteínas Nucleares/fisiologia , Plasmídeos , Recombinação Genética , Replicação Viral/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Ágar , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , RNA Interferente Pequeno
20.
EMBO Rep ; 7(7): 716-21, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16799465

RESUMO

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein-Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits.


Assuntos
Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Linhagem Celular , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/genética , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Origem de Replicação , Telômero/genética , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas
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