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1.
Nat Microbiol ; 3(9): 1075, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29679064

RESUMEN

In the version of this Letter originally published, the authors reported on the use of 2,5-dimethylpyrrolyl benzoic acid to block Ephrin receptors. In 2011, it was reported that newly synthesized 2,5-dimethylpyrrolyl benzoic acid lacked the previously reported EphA2 antagonizing activity1. However, the purchased compound did in fact have the activity initially reported, suggesting that an uncharacterized alteration occurred during storage. The authors therefore wish to clarify that the compound used in their study should be more accurately referred to as a 2,5-dimethylpyrrolyl benzoic acid derivative. All references to 2,5-dimethylpyrrolyl benzoic acid in the Letter have now been changed to reflect this.Although 2,5-dimethylpyrrolyl benzoic acid derivatives have been reported to have off-target effects2, as do most small-molecule inhibitors, the multiple complementary methods and techniques used demonstrate that EphA2 is a key Epstein-Barr virus epithelial cell receptor. The conclusions of the study are therefore unchanged.

2.
Nat Microbiol ; 3(2): 1-8, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29292383

RESUMEN

Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR-Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi's sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.


Asunto(s)
Efrina-A2/metabolismo , Células Epiteliales/virología , Herpesvirus Humano 4/patogenicidad , Receptor EphA2/metabolismo , Internalización del Virus , Animales , Benzoatos/antagonistas & inhibidores , Células CHO , Sistemas CRISPR-Cas , Cricetulus , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Glicoproteínas de Membrana , Chaperonas Moleculares , Interferencia de ARN , Receptor EphA2/efectos de los fármacos , Receptor EphA2/genética , Proteínas Virales
3.
Cell Host Microbe ; 22(4): 561-573.e4, 2017 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-29024646

RESUMEN

Epstein-Barr virus (EBV) transforms B cells to continuously proliferating lymphoblastoid cell lines (LCLs), which represent an experimental model for EBV-associated cancers. EBV nuclear antigens (EBNAs) and LMP1 are EBV transcriptional regulators that are essential for LCL establishment, proliferation, and survival. Starting with the 3D genome organization map of LCL, we constructed a comprehensive EBV regulome encompassing 1,992 viral/cellular genes and enhancers. Approximately 30% of genes essential for LCL growth were linked to EBV enhancers. Deleting EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets included MCL1, IRF4, and EBF. MYC ESE looping to the transcriptional stat site of MYC was dependent on EBNAs. Deleting MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study provides a comprehensive view of the spatial organization of chromatin during EBV-driven cellular transformation.


Asunto(s)
Linfocitos B/virología , Cromatina/virología , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno , Línea Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo
4.
Proc Natl Acad Sci U S A ; 114(36): 9683-9688, 2017 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-28831010

RESUMEN

Nasopharyngeal carcinoma (NPC) most frequently occurs in southern China and southeast Asia. Epidemiology studies link NPC to genetic predisposition, Epstein-Barr virus (EBV) infection, and environmental factors. Genetic studies indicate that mutations in chromatin-modifying enzymes are the most frequent genetic alterations in NPC. Here, we used H3K27ac chromatin immune precipitation followed by deep sequencing (ChIP-seq) to define the NPC epigenome in primary NPC biopsies, NPC xenografts, and an NPC cell line, and compared them to immortalized normal nasopharyngeal or oral epithelial cells. We identified NPC-specific enhancers and found these enhancers were enriched with nuclear factor κB (NF-κB), IFN-responsive factor 1 (IRF1) and IRF2, and ETS family members ETS1 motifs. Normal cell-specific enhancers were enriched with basic leucine zipper family members and TP53 motifs. NPC super-enhancers with extraordinarily broad and high H3K27ac signals were also identified, and they were linked to genes important for oncogenesis including ETV6. ETV6 was also highly expressed in NPC biopsies by immunohistochemistry. High ETV6 expression correlated with a poor prognosis. Furthermore, we defined the EBV episome epigenetic landscapes in primary NPC tissue.


Asunto(s)
Carcinoma/genética , Elementos de Facilitación Genéticos , Neoplasias Nasofaríngeas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Adolescente , Adulto , Anciano , Animales , Azepinas/farmacología , Carcinoma/etiología , Carcinoma/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Elementos de Facilitación Genéticos/efectos de los fármacos , Epigénesis Genética , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Genoma Viral , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Código de Histonas/genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/etiología , Neoplasias Nasofaríngeas/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Pronóstico , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Adulto Joven , Proteína ETS de Variante de Translocación 6
5.
Cell Rep ; 19(7): 1479-1493, 2017 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-28514666

RESUMEN

Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.


Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/virología , Herpesvirus Humano 4/fisiología , Proteómica/métodos , Replicación Viral , Ciclo Celular , Membrana Celular/metabolismo , Proteínas del Sistema Complemento/metabolismo , Regulación hacia Abajo , Humanos , Proteolisis , Receptores de Antígenos de Linfocitos B/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Regulación hacia Arriba
6.
Proc Natl Acad Sci U S A ; 114(18): 4751-4756, 2017 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-28351978

RESUMEN

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/inmunología , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Humano 4/inmunología , Enfermedad de Hodgkin/inmunología , Linfoma de Células B/inmunología , Neoplasias Experimentales/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Centro Germinal/inmunología , Centro Germinal/patología , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Ratones Mutantes , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas de la Matriz Viral/genética
7.
Proc Natl Acad Sci U S A ; 113(49): 14121-14126, 2016 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-27864512

RESUMEN

Epstein-Barr virus (EBV) super-enhancers (ESEs) are essential for lymphoblastoid cell (LCL) growth and survival. Reanalyses of LCL global run-on sequencing (Gro-seq) data found abundant enhancer RNAs (eRNAs) being transcribed at ESEs. Inactivation of ESE components, EBV nuclear antigen 2 (EBNA2) and bromodomain-containing protein 4 (BRD4), significantly decreased eRNAs at ESEs -428 and -525 kb upstream of the MYC oncogene transcription start site (TSS). shRNA knockdown of the MYC -428 and -525 ESE eRNA caused LCL growth arrest and reduced cell growth. Furthermore, MYC ESE eRNA knockdown also significantly reduced MYC expression, ESE H3K27ac signals, and MYC ESEs looping to MYC TSS. These data indicate that ESE eRNAs strongly affect cell gene expression and enable LCL growth.


Asunto(s)
Elementos de Facilitación Genéticos , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Trastornos Linfoproliferativos/virología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Endonucleasas , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo
8.
Front Med ; 10(1): 61-75, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26969667

RESUMEN

The latent expression pattern of Epstein-Barr Virus (EBV) genes in nasopharyngeal carcinoma (NPC) has been extensively investigated, and the expression of several lytic genes in NPC has been reported. However, comprehensive information through EBV transcriptome analysis in NPC is limited. We performed paired-end RNA-seq to systematically and comprehensively characterize the expression of EBV genes in NPC tissue and C666-1 NPC cell line, which consistently carries EBV. In addition to the transcripts restricted to type II latency infection, the type III latency EBNA3s genes and a substantial number of lytic genes, such as BZLF1, BRLF1, and BMRF1, were detected through RNA-seq and were further verified in C666-1 cells and NPC tissue through realtime PCR.We also performed clustering analysis to classify NPC patient groups in terms of EBV gene expression, which presented two subtypes of NPC samples. Results revealed interesting patterns of EBV gene expression in NPC patients. This clustering was correlated with many signaling pathways, such as those related to heterotrimeric G-protein signaling, inflammation mediated by chemokine and cytokine signaling, ribosomes, protein metabolism, influenza infection, and ECM-receptor interaction. Our combined findings suggested that the expression of EBV genes in NPC is restricted not only to type II latency genes but also to type III latency and lytic genes. This study provided further insights into the potential role of EBV in the development of NPC.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virología , Línea Celular Tumoral , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Transactivadores/metabolismo , Latencia del Virus/genética
9.
Proc Natl Acad Sci U S A ; 113(8): 2229-34, 2016 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-26858444

RESUMEN

Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.


Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Ribosómicas/metabolismo , Linfocitos B/metabolismo , Linfocitos B/virología , Línea Celular , ADN Viral/genética , ADN Viral/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Técnicas de Silenciamiento del Gen , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Origen de Réplica , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Activación Transcripcional , Nucleolina
10.
Proc Natl Acad Sci U S A ; 112(37): 11612-7, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26305967

RESUMEN

Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.


Asunto(s)
Centro Germinal/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Autoanticuerpos/química , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/virología , Diferenciación Celular , Linaje de la Célula , Cruzamientos Genéticos , Epigénesis Genética , Citometría de Flujo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Bazo/citología , Dedos de Zinc
11.
Proc Natl Acad Sci U S A ; 112(35): 11036-41, 2015 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-26290577

RESUMEN

EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection.


Asunto(s)
Infecciones por Virus de Epstein-Barr/fisiopatología , Cadenas Pesadas de Miosina/fisiología , Nasofaringe/virología , Secuencia de Aminoácidos , Línea Celular Transformada , Humanos , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Nasofaringe/patología
12.
J Extracell Vesicles ; 4: 26334, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25865256

RESUMEN

The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1-TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1-TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1-TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1-TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that "signalling endosomes" in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

13.
Cell Host Microbe ; 17(2): 205-16, 2015 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-25639793

RESUMEN

Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.


Asunto(s)
Linfocitos B/virología , Proliferación Celular , Regulación de la Expresión Génica , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Proteínas Oncogénicas/metabolismo , Proteínas Virales/metabolismo , Linfocitos B/fisiología , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Transcripción Genética
14.
Nat Commun ; 6: 6240, 2015 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-25670642

RESUMEN

Epstein-Barr virus (EBV) is implicated as an aetiological factor in B lymphomas and nasopharyngeal carcinoma. The mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells remain elusive. EBV glycoprotein B (gB) is the critical fusion protein for infection of both B and epithelial cells, and determines EBV susceptibility of non-B cells. Here we show that neuropilin 1 (NRP1) directly interacts with EBV gB(23-431). Either knockdown of NRP1 or pretreatment of EBV with soluble NRP1 suppresses EBV infection. Upregulation of NRP1 by overexpression or EGF treatment enhances EBV infection. However, NRP2, the homologue of NRP1, impairs EBV infection. EBV enters nasopharyngeal epithelial cells through NRP1-facilitated internalization and fusion, and through macropinocytosis and lipid raft-dependent endocytosis. NRP1 partially mediates EBV-activated EGFR/RAS/ERK signalling, and NRP1-dependent receptor tyrosine kinase (RTK) signalling promotes EBV infection. Taken together, NRP1 is identified as an EBV entry factor that cooperatively activates RTK signalling, which subsequently promotes EBV infection in nasopharyngeal epithelial cells.


Asunto(s)
Células Epiteliales/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Neoplasias Nasofaríngeas/virología , Neuropilina-1/metabolismo , Internalización del Virus , Carcinoma , Línea Celular Tumoral , Endocitosis , Células Epiteliales/patología , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Humanos , Fusión de Membrana , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Neuropilina-2/metabolismo , Unión Proteica , Transporte de Proteínas , Transducción de Señal
15.
Exp Mol Med ; 47: e131, 2015 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-25613728

RESUMEN

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.


Asunto(s)
Genes Virales , Herpesvirus Humano 4/fisiología , Latencia del Virus , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , MicroARNs/genética , Neoplasias/etiología , Unión Proteica , ARN Viral/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo
16.
Proc Natl Acad Sci U S A ; 112(2): 554-9, 2015 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-25540416

RESUMEN

Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN/genética , ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Linfocitos B/metabolismo , Linfocitos B/virología , Sitios de Unión/genética , Línea Celular , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Ciclina D2/genética , Elementos de Facilitación Genéticos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genes bcl-2 , Genes myc , Genes p16 , Genoma Humano , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno/genética , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Factores Reguladores del Interferón/metabolismo , Regiones Promotoras Genéticas , Proteínas Virales/genética , Proteínas Virales/metabolismo
17.
Cell Rep ; 8(5): 1595-606, 2014 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-25159142

RESUMEN

The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade.


Asunto(s)
Linfocitos B/metabolismo , Elementos de Facilitación Genéticos , Redes Reguladoras de Genes , Genoma Humano , FN-kappa B/metabolismo , Línea Celular Tumoral , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/genética , Regiones Promotoras Genéticas , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Activación Transcripcional
18.
Cell Signal ; 26(12): 2730-7, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25134449

RESUMEN

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein-Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.


Asunto(s)
FN-kappa B/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Regulación hacia Abajo/fisiología , Células HEK293 , Humanos , Interleucina-8/metabolismo , Dominios RING Finger/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Proteínas de la Matriz Viral
19.
Antiviral Res ; 104: 73-83, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24486954

RESUMEN

The replication and persistence of extra chromosomal Epstein-Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt's lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo.


Asunto(s)
Antivirales/farmacología , Replicación del ADN/efectos de los fármacos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genoma Viral , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Replicación Viral/efectos de los fármacos , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos
20.
Mol Cell Biol ; 34(3): 325-34, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24248603

RESUMEN

We have previously reported that interleukin-1 (IL-1) receptor-associated kinase (IRAK1) is essential for Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced p65/RelA serine 536 phosphorylation and NF-κB activation but not for IκB kinase α (IKKα) or IKKß activation (Y. J. Song, K. Y. Jen, V. Soni, E. Kieff, and E. Cahir-McFarland, Proc. Natl. Acad. Sci. U. S. A. 103:2689-2694, 2006, doi:10.1073/pnas.0511096103). Since the kinase activity of IRAK1 is not required for LMP1-induced NF-κB activation, IRAK1 is proposed to function as a scaffold protein to recruit a p65/RelA serine 536 kinase(s) to enhance NF-κB-dependent transcriptional activity. We now report that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with IRAK1 and is critical for LMP1-induced p65/RelA serine 536 phosphorylation and NF-κB activation. CaMKII bound the death domain of IRAK1 and directly phosphorylated p65/RelA at serine 536 in vitro. Downregulation of CaMKII activity or expression significantly reduced LMP1-induced p65/RelA serine 536 phosphorylation and NF-κB activation. Furthermore, LMP1-induced CaMKII activation and p65/RelA serine 536 phosphorylation were significantly reduced in IRAK1 knockout (KO) mouse embryonic fibroblasts (MEFs). Thus, IRAK1 may recruit and activate CaMKII, which phosphorylates p65/RelA serine 536 to enhance the transactivation potential of NF-κB in LMP1-induced NF-κB activation pathway.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Células Cultivadas , Embrión de Mamíferos/citología , Activación Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Ratones , Ratones Noqueados , Mutación , FN-kappa B/genética , Fosforilación , Unión Proteica , Interferencia de ARN , Serina/genética , Serina/metabolismo , Factor de Transcripción ReIA/genética , Proteínas de la Matriz Viral/genética
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