RESUMEN
Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.
Asunto(s)
Antígenos Virales/química , Linfocitos B/virología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Factores Reguladores del Interferón/química , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/metabolismo , Transactivadores/química , Secuencias de Aminoácidos , Linfocitos B/citología , Sitios de Unión , Proliferación Celular , Inmunoprecipitación de Cromatina , Antígenos Nucleares del Virus de Epstein-Barr , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Histonas/química , Humanos , Linfoma/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3 , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA3A are each essential for EBV conversion of primary human B lymphocytes into continuously proliferating lymphoblast cell lines (LCLs) and for maintaining LCL growth. We now find that EBNA3C and EBNA3A's essential roles are to repress p16(INK4A) and p14(ARF). In the absence of EBNA3C or EBNA3A, p16(INK4A) and p14(ARF) expression increased and cell growth ceased. EBNA3C inactivation did not alter p16(INK4A) promoter CpG methylation, but reduced already low H3K27me3, relative to resting B cells, and increased H3K4me3 and H3-acetylation, linking EBNA3C inactivation to histone modifications associated with increased transcription. Importantly, knockdown of p16(INK4A) or p14(ARF) partially rescued LCLs from EBNA3C or EBNA3A inactivation-induced growth arrest and knockdown of both rescued LCL growth, confirming central roles for p16(INK4A) and p14(ARF) in LCL growth arrest following EBNA3C or EBNA3A inactivation. Moreover, blockade of p16(INK4A) and p14(ARF) effects on pRb and p53 by human papilloma virus type 16 E7 and E6 expression, sustained LCL growth after EBNA3C or EBNA3A inactivation. These data indicate that EBNA3C and EBNA3A joint repression of CDKN2A p16(INK4A) and p14(ARF) is essential for LCL growth.
Asunto(s)
División Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Proteína p14ARF Supresora de Tumor/fisiología , Línea Celular , Islas de CpG , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4).
Asunto(s)
Antígenos Virales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Genes/genética , Factores de Transcripción/metabolismo , Análisis de Varianza , Teorema de Bayes , Línea Celular Tumoral , Análisis por Conglomerados , Antígenos Nucleares del Virus de Epstein-Barr , Perfilación de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Activación de Linfocitos , Análisis por Micromatrices , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Epstein-Barr virus (EBV) establishes lifelong latency in B-lymphocytes following infection. Although in immune-competent individuals EBV remains in a quiescent state, in immunodeficient individuals, such as those with AIDS or transplant recipients, B-lymphocytes infected with EBV proliferate to give rise to lymphoproliferative diseases. Similarly, in vitro, EBV transforms human B-lymphocytes into indefinitely growing lymphoblastoid cell lines (LCLs) in the absence of cytotoxic T-lymphocytes. Although LCLs harbor the entire EBV genome as an episome, in most cells the virus remains in a latent state expressing a fraction of EBV genes, and lytic infection occurs spontaneously but only in a small percentage of cells. Here, we report that lytic infection contributes to EBV-induced lymphoproliferation by a paracrine mechanism. An EBV immediate-early protein, BZLF1, induces IL-13, thus facilitating the proliferation of EBV-transformed B-lymphocytes in the presence of T-lymphocytes. These data suggest that lytic gene products could contribute to virus-induced oncogenesis by a paracrine mechanism.
Asunto(s)
Linfocitos B/virología , Transformación Celular Viral , Herpesvirus Humano 4/fisiología , Linfocitos T/inmunología , Linfocitos B/metabolismo , Línea Celular , Transformación Celular Viral/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Interleucina-13/metabolismo , Comunicación Paracrina , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de la Matriz Viral/metabolismoRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for efficient conversion of primary human B lymphocytes to lymphoblastoid cell lines (LCLs) and for continued LCL growth. We used a transcomplementation assay in the context of LCLs transformed by an EBV with a conditional EBNA3C to identify the EBNA3C amino acids (aa) necessary for maintaining LCL growth. Surprisingly, we found that most EBNA3C aa were essential for continued LCL growth. Only EBNA3C mutants deleted for residues within aa 507-515, 516-620, 637-675, or 676-727 maintained full LCL growth, and EBNA3C mutants deleted for residues within aa 728-732 or 910-992 maintained slow LCL growth. In contrast, EBNA3C lacking aa 180-231, which mediate RBP-Jkappa association and are necessary for EBNA3C abrogation of EBNA2-induced transcription through RBP-Jkappa, could not support LCL growth. Furthermore, 2 EBNA3C alanine substitution mutants within aa 180-231, which were wild-type (wt) in abrogating EBNA2-mediated transcription through RBP-Jkappa, maintained LCL growth, and 2 alanine substitution mutants within aa 180-231, which were null in abrogating EBNA2-mediated transcription through RBP-Jkappa, did not maintain LCL growth. This indicates that EBNA3C regulation of transcription through RBP-Jkappa is critical to maintaining LCL growth. Several other EBNA3C functions also are critical for LCL growth, because EBNA3C mutants deleted for residues within aa 130-159, 251-506, or 733-909 were wt in abrogating transcription through RBP-Jkappa and expression level, but did not maintain LCL growth.
Asunto(s)
Antígenos Virales/fisiología , Linfocitos B/virología , Transformación Celular Viral , Herpesvirus Humano 4/patogenicidad , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Linfoma de Células B/virología , Antígenos Virales/genética , Antígenos Nucleares del Virus de Epstein-Barr , Regulación de la Expresión Génica , Prueba de Complementación Genética , Humanos , Linfoma de Células B/patología , Mutagénesis Sitio-Dirigida , Mutación , Transcripción Genética , Proteínas ViralesRESUMEN
Burkitt lymphoma (BL) is etiologically associated with Epstein-Barr virus (EBV). EBV-positive BL tumors display two latent forms of infection. One is referred to as latency I infection, in which EBV expresses the virus genome maintenance protein EBNA1 as the only viral protein. The other is referred to as Wp-restricted latency and was recently identified in a subset of BL tumors. In these tumors, EBV expresses EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the viral Bcl-2 homologue BHRF1, all of which are driven by the BamHI W promoter (Wp). To investigate the role of EBV in Wp-restricted BL, we conditionally expressed a dominant-negative EBNA1 (dnEBNA1) mutant which interrupts the virus genome maintenance function of EBNA1 in the P3HR-1 BL cell line. Induction of dnEBNA1 expression caused loss of the EBV genome and resulted in apoptosis of P3HR-1 cells in the absence of exogenous apoptosis inducers, indicating that P3HR-1 cells cannot survive without EBV. Stable transfection of the BHRF1 gene into P3HR-1 cells rescued the cells from the apoptosis induced by dnEBNA1 expression, whereas stable transfection of truncated EBNA-LP, EBNA3A, or EBNA3C did not. Moreover, knockdown of BHRF1 expression in P3HR-1 cells resulted in increased cell death. These results indicate that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor. Our finding implies a critical contribution of BHRF1 to the pathogenesis of Wp-restricted BLs.
Asunto(s)
Linfoma de Burkitt/metabolismo , Supervivencia Celular/fisiología , Herpesvirus Humano 4/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Virales/metabolismo , Animales , Apoptosis/genética , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Virales/genética , Latencia del Virus/genéticaRESUMEN
B lymphocytes converted into lymphoblastoid cell lines (LCLs) by an Epstein-Barr virus that expresses a conditional EBNA3C require complementation with EBNA3C for growth under nonpermissive conditions. Complementation with relatively large EBNA3C deletion mutants identified amino acids (aa) 1 to 506 (which includes the RBP-Jkappa/CSL [RBP-Jkappa] binding domain) and 733 to 909 to be essential for LCL growth, aa 728 to 732 and 910 to 992 to be important for full wild-type (wt) growth, and only aa 507 to 727 to be unimportant (S. Maruo, Y. Wu, T. Ito, T. Kanda, E. D. Kieff, and K. Takada, Proc. Natl. Acad. Sci. USA 106:4419-4424, 2009). When mutants with smaller deletions were used, only aa 51 to 400 and 851 to 900 were essential for LCL growth; aa 447 to 544, 701 to 750, 801 to 850, and 901 to 992 were important for full wt growth; and aa 4 to 50, 401 to 450, 550 to 707, and 751 to 800 were unimportant. These data reduce the EBNA3C essential residues from 68% to 40% of the open reading frame. Point mutations confirmed RBP-Jkappa binding to be essential for wt growth and indicated that SUMO and CtBP binding interactions were important only for full wt growth. EBNA3C aa 51 to 150, 249 to 311, and 851 to 900 were necessary for maintaining LCL growth, but not RBP-Jkappa interaction, and likely mediate interactions with other key cell proteins. Moreover, all mutants null for LCL growth had fewer S+G(2)/M-phase cells at 14 days, consistent with EBNA3C interaction with RBP-Jkappa as well as aa 51 to 150, 249 to 311, and 851 to 900 being required to suppress p16(INK4A) (S. Maruo, Y. Wu, S. Ishikawa, T. Kanda, D. Iwakiri, and K. Takada, Proc. Natl. Acad. Sci. USA 103:19500-19505, 2006). We have confirmed that EBNA3C upregulates TCL1 and discovered that EBNA3C upregulates TCL1 through RBP-Jkappa, indicating a central role for EBNA3C interaction with RBP-Jkappa in mediating cell gene transcription.
Asunto(s)
Antígenos Virales/fisiología , Transformación Celular Neoplásica , Herpesvirus Humano 4/patogenicidad , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Antígenos Virales/genética , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Eliminación de Secuencia , Regulación hacia ArribaRESUMEN
The Epstein-Barr virus (EBV) immediate-early transactivator BZLF1 plays a key role in switching EBV infection from the latent to the lytic form by stimulating the expression cascade of lytic genes; it also regulates the expression of several cellular genes. Recently, we reported that BZLF1 is expressed in primary human B cells early after EBV infection. To investigate whether this BZLF1 expression early after infection plays a role in the EBV-induced growth transformation of primary B cells, we generated BZLF1-knockout EBV and quantitatively evaluated its transforming ability compared with that of wild-type EBV. We found that the 50% transforming dose of BZLF1-knockout EBV was quite similar to that of wild-type EBV. Established lymphoblastoid cell lines (LCLs) harbouring BZLF1-knockout EBV were indistinguishable from LCLs harbouring wild-type EBV in their pattern of latent gene expression and in their growth in vitro. Furthermore, the copy numbers of EBV episomes were very similar in the LCLs harbouring BZLF1-knockout EBV and in those harbouring wild-type EBV. These data indicate that disrupting BZLF1 expression in the context of the EBV genome, and the resultant inability to enter lytic replication, have little impact on the growth of LCLs and the steady-state copy number of EBV episomes in established LCLs.
Asunto(s)
Linfocitos B/fisiología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Activación de Linfocitos/fisiología , Transactivadores/metabolismo , Línea Celular Tumoral , Eliminación de Gen , Regulación Viral de la Expresión Génica/fisiología , Humanos , Plásmidos , Latencia del VirusRESUMEN
Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95-8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12-15 days through 24 days post-infection in tissue models infected with B95-8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP(+) cells were CD3(-) CD56(-) CD19(+) HLA-DR(+), and represented both naïve (immunoglobulin D(+)) and memory (CD27(+)) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Herpesvirus Humano 4/fisiología , Tonsila Palatina/citología , Tonsila Palatina/virología , Replicación Viral , Aciclovir/farmacología , Antivirales/farmacología , Separación Celular , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes/genética , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/genética , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/virología , Tonsila Palatina/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Epstein-Barr virus (EBV)-encoded RNA 1 (EBER1) and EBER2 are untranslated RNAs and the most abundant viral transcripts in latently EBV-infected cells. We previously reported that EBERs play a critical role in efficient EBV-induced growth transformation of primary B cells. To investigate whether EBER1 and EBER2 have distinct roles in B-cell growth transformation, recombinant EBVs carrying either EBER1 or EBER2 were generated. The transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Lymphoblastoid cell lines (LCLs) established with EBVs carrying EBER2 proliferated at low cell densities, while LCLs established with EBVs carrying EBER1 did not. Interleukin 6 (IL-6) production in LCLs expressing EBER2 was more abundant than in those lacking EBER2. The growth of LCLs lacking EBER2 was enhanced by the addition of recombinant IL-6 to the cell culture, while the growth of EBER2-expressing LCLs was inhibited by a neutralizing anti-IL-6 antibody. These results demonstrate that EBER2, but not EBER1, contributes to efficient B-cell growth transformation. We conclude that EBER1 and EBER2, despite their structural similarity, have different functions in latently infected lymphoblastoid cells.
Asunto(s)
Linfocitos B/virología , Transformación Celular Viral , Herpesvirus Humano 4/patogenicidad , ARN Viral/fisiología , Linfocitos B/patología , Línea Celular , Proliferación Celular , Humanos , Interleucina-6/biosíntesis , Interleucina-6/farmacologíaRESUMEN
In eukaryotes, many latent viruses replicate as extrachromosomal molecules, called episomes, and efficiently segregate to daughter cells by noncovalently attaching to mitotic chromosomes. To understand the mechanism governing the processes, we analyzed the detailed subcellular localization of Epstein-Barr virus (EBV) genomes and a viral protein EBNA1, a bridging molecule between viral genomes and cellular chromatin. In the cells that were infected with a recombinant EBV expressing epitope-tagged EBNA1, EBNA1 localized to intranuclear punctate dots, which coincided with the localization of EBV genomes as revealed by fluorescence in situ hybridization (FISH). A significant number of EBNA1 dots were found to localize symmetrically on sister chromatids of mitotic chromosomes. Such symmetrical localization of EBNA1 dots was observed in prematurely condensed G2 chromosomes as well, correlating with the presence of closely spaced double dots of EBNA1 in G2-phase-enriched cells. The EBNA1 double dots were occasionally interconnected by the FISH signals of EBV episomes, exhibiting a dumbbell-like appearance. Thus, we propose that the partitioning of EBNA1 molecules onto sister chromatids during cellular DNA replication underlies the non-stochastic segregation of extrachromosomally replicating viral genomes.
Asunto(s)
Cromátides/genética , Replicación del ADN/genética , Genoma Viral , Herpesvirus Humano 4/genética , Plásmidos/genética , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/genética , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Dicetopiperazinas , Inhibidores Enzimáticos/farmacología , Antígenos Nucleares del Virus de Epstein-Barr/análisis , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Fase G2/genética , Dosificación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hidroxiurea/farmacología , Hibridación Fluorescente in Situ , Toxinas Marinas , Mitosis/efectos de los fármacos , Modelos Genéticos , Oxazoles/farmacología , Piperazinas/farmacología , Plásmidos/análisis , Inhibidores de Topoisomerasa IIRESUMEN
We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.
Asunto(s)
Linfocitos B/virología , Proteínas de Unión al ADN/genética , Genes Inmediatos-Precoces , Herpesvirus Humano 4/patogenicidad , Transactivadores/genética , Proteínas Virales/genética , Latencia del Virus , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/fisiología , Humanos , Transactivadores/metabolismo , Proteínas Virales/metabolismoRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) association with RBP-Jkappa is essential for regulation of virus and cell gene transcription and B lymphocyte transformation into infinitely proliferating lymphoblastoid cells (LCLs). To identify EBNA2-regulated cell genes in LCLs, an EBV recombinant that expresses EBNA2 with its C terminus fused in frame to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor (E2HTF) was used to transform primary B lymphocytes to LCLs. In the presence of 4HT, E2HTF expression level and effects on the LMP1 promoter in transfected BJAB lymphoblasts were similar to EBNA2. In 4HT-supplemented medium, E2HTF EBV recombinant infected LCLs were also similar to EBNA2 LCLs in outgrowth but required higher serum and a restricted range of cell concentrations for consistent growth. In medium without 4HT, E2HTF localized to the cytoplasm, c-myc levels substantially decreased within 6 h, cells stopped growing, and levels of other EBNAs and LMP1 remained stable for 24 h. Over this 24-h period, 30 cell RNAs decreased 2-fold, and 51 other RNAs decreased 1.5-fold. These RNAs encode proteins important in cell adhesion or signaling, transcription, RNA processing, cell-cycle regulation, and survival. Real-time RT-PCR confirmed EBNA2-dependent expression of eight RNAs.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Linfocitos/metabolismo , Linfocitos/patología , ARN/biosíntesis , ARN/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Computadores , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/genética , Humanos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Notch1/metabolismo , Receptores de IgE/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
Epstein-Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C-HT. In the presence of 4HT, LCLs expressed the E3C-HT protein and grew like WT LCLs. When E3C-HT EBV-infected LCLs were transferred to medium without 4HT, E3C-HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C-HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.
Asunto(s)
Linfocitos B/virología , Ciclo Celular/fisiología , Proliferación Celular , Transformación Celular Viral/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Linfocitos B/citología , Western Blotting , Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Cartilla de ADN , Citometría de Flujo , Prueba de Complementación Genética , Humanos , Inmunoprecipitación , Proteína de Retinoblastoma/metabolismo , Tamoxifeno/análogos & derivadosRESUMEN
Experimental reverse genetic replacement of Epstein-Barr virus nuclear antigen 3A (EBNA3A) with a conditional mutant EBNA3A revealed that EBNA3A is critical for continued lymphoblastoid cell (LCL) growth. Wild-type (wt) EBNA3A expressed in the LCLs specifically sustained growth under nonpermissive conditions, whereas EBNA3B or EBNA3C expression had no effect (S. Mauro, E. Johannsen, D. Illanes, A. Cooper, and E. Kieff, J. Virol. 77:10437-10447, 2003). This genetic system and related biochemical assays have now been used to discover that EBNA3A lacking amino acid residues 170 to 240 (delta170-240), TLGC202 to AAGA202, or delta300-386, which are deficient in repression of EBNA2 activation of an RBP-Jkappa/CBF1-dependent EBV Cp enhancer, are null mutations for LCL growth, whereas EBNA3A delta2-124, delta410-439, delta440-470, delta470-500, delta500-523, delta523-612, and delta620-820, which are wt in repression are wt for LCL growth. Thus, EBNA3A regulation of transcription through RBP-Jkappa/CBF1 is critical for LCL growth. EBNA3A mutants deleted of amino acid residues 240 to 300, 386 to 410, or 827 to 944 were intermediate, null, or intermediate, respectively, for LCL growth despite being wt for RBP-Jkappa association and repression. Amino acid residues 240 to 300, 386 to 410, and, particularly, C-terminal residues 827 to 944 are therefore likely to contribute to LCL growth through RBP-Jkappa-independent mechanisms.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/química , Linfocitos/fisiología , Proteínas Nucleares/fisiología , Transcripción Genética , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteínas ViralesRESUMEN
Epstein-Barr virus (EBV) is associated with various epithelial malignancies such as nasopharyngeal carcinoma and gastric carcinoma, and causes oral hairy leukoplakia, a productive EBV infection of the differentiated epithelium of the tongue. However, it is not clear by what mechanism EBV infects epithelial cells. We generated a recombinant EBV that expresses enhanced green fluorescent protein in order to monitor EBV entrance into epithelial cells quickly and quantitatively. Using this monitoring system, we examined the roles of gp350 and gp25 in EBV infection of epithelial cells by utilizing soluble forms of the gp350 and gp25 proteins. EBV infection of three of four examined epithelial cell lines, 293, NU-GC-3 and Lovo, was almost completely blocked by pretreatment of cells with a soluble form of gp350 (designated gp350Ig), and this blockage was dependent on the CD21-binding region of gp350. On the other hand, infection of the other epithelial cell line, AGS, was not inhibited at all by pretreatment with gp350Ig. Moreover, we found that a soluble form of gp25 (designated gp25Ig) preferentially bound to epithelial cells rather than B cells, and pretreatment of cells with gp25Ig substantially blocked EBV infection of some epithelial cells. These results indicate the existence of two distinct pathways in EBV infection of epithelial cells, a gp350-dependent pathway and a gp350-independent pathway, and that gp25 can play a role in the infection of some epithelial cells.
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Células Epiteliales/virología , Herpesvirus Humano 4/fisiología , Proteínas Virales/fisiología , Animales , Linfocitos B/metabolismo , Células COS , Humanos , Proteínas Recombinantes de Fusión/fisiología , Células Tumorales CultivadasRESUMEN
To quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.
Asunto(s)
Linfocitos B/patología , Transformación Celular Viral , Herpesvirus Humano 4/fisiología , Proteínas de la Matriz Viral/metabolismo , Activación Viral , Antígenos Virales/genética , Antígenos Virales/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/virología , Western Blotting , Calcio/metabolismo , Señalización del Calcio , Línea Celular Transformada , Supervivencia Celular , Exones/genética , Eliminación de Gen , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Agregación de Receptores , Receptores de Antígenos de Linfocitos B/inmunología , Transfección , Proteínas de la Matriz Viral/genética , Latencia del Virus , Replicación ViralRESUMEN
To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.
Asunto(s)
Linfocitos B/fisiología , Linfocitos B/virología , División Celular/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/genética , Tamoxifeno/análogos & derivados , Humanos , Proteínas Proto-Oncogénicas c-myc/análisis , Receptores de Complemento 3d/análisis , Receptores de IgE/análisis , Recombinación Genética , Tamoxifeno/farmacología , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/análisisRESUMEN
Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jkappa/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jkappa, did not change EBNA3C association with RBP-Jkappa or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jkappa, decreased c-myc expression, and caused G(0)/G(1) growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jkappa-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jkappa binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jkappa binding domain. The dependence of EBNA3A effects on the core RBP-Jkappa interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jkappa association strongly implicates RBP-Jkappa in c-myc promoter regulation.