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1.
J Virol ; 89(10): 5222-37, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25787276

RESUMO

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


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Variação Genética , Genoma Viral , Herpesvirus Humano 4/genética , Sequência de Aminoácidos , Antígenos Virais/genética , Portador Sadio/virologia , Linhagem Celular Tumoral , DNA Viral/genética , Epitopos de Linfócito T/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/classificação , Herpesvirus Humano 4/isolamento & purificação , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Proteínas da Matriz Viral/genética
2.
J Pathol ; 231(3): 367-77, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23934731

RESUMO

Nasopharyngeal carcinoma (NPC) is a cancer common in southern China and South East Asia that is causally linked to Epstein-Barr virus (EBV) infection. Here, we demonstrate that NPC displays frequent dysregulation of the Hedgehog (HH) pathway, a pathway implicated in the maintenance of stem cells, but whose aberrant activation in adult tissues can lead to cancer. Using authentic EBV-positive carcinoma-derived cell lines and nasopharyngeal epithelial cell lines latently infected with EBV as models for NPC in vitro, we show that EBV activates the HH signalling pathway through autocrine induction of SHH ligand. Moreover, we find that constitutive engagement of the HH pathway induces the expression of a number of stemness-associated genes and imposes stem-like characteristics on EBV-infected epithelial cells in vitro. Using epithelial cells expressing individual EBV latent genes detected in NPC, we show that EBNA1, LMP1, and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. Our findings not only identify a role for dysregulated HH signalling in NPC oncogenesis, but also provide a novel rationale for therapeutic intervention.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/virologia , Proteínas Hedgehog/metabolismo , Herpesvirus Humano 4/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/virologia , Transdução de Sinais , Carcinoma , Linhagem Celular , Transformação Celular Viral , Células Epiteliais/patologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , Ligantes , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/patologia , Fenótipo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
3.
Int J Cancer ; 131(4): E348-61, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21913186

RESUMO

Gene expression profiling has recently enabled the reclassification of aggressive non-Hodgkin lymphomas (aNHL) into distinct subgroups. In Burkitt lymphoma (BL) aberrant c-Myc activity results from IG-MYC translocations. However, MYC aberrations are not limited to BLs and then have a negative prognostic impact. In this study, we investigated to which extent aberrant c-Myc activity plays a functional role in other aNHL and whether it is independent from MYC translocations. Based on a combined microarray analysis of human germinal center (GC) B cells transfected with c-Myc and 220 aNHLs cases, we developed a "c-Myc index." This index measures the extent to which lymphomas express c-Myc responsive genes. It comprises genes that are affected in a variety of tumors compared to normal tissue. This supports the view that aberrant c-Myc expression in GC B cells triggers a tumor-like expression pattern. As expected, the "c-Myc index" is very high in molecular Burkitt lymphoma (mBL), but more importantly also high within other aNHL. It constitutes a negative prognostic marker independent of established risk factors and of the presence of a MYC translocation. Our data provide new insights into the role of c-Myc activity in different lymphomas and raises the question of treatment changes for those patients under risk.


Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Primers do DNA , Imunofluorescência , Humanos , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Reação em Cadeia da Polimerase em Tempo Real
4.
Bioorg Med Chem Lett ; 22(1): 129-32, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22169264

RESUMO

Single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer were identified using oligodeoxynucleotide probe sequences bearing internal anthracene fluorophores proximal to the SNP site. Depending upon the nature of the synthesised target sequences, probe-target duplex formation could lead to enhanced or attenuated fluorescence emission from the anthracene, enabling detection of a proximal base-pair as either matching or mismatching.


Assuntos
Sondas de DNA/química , DNA/química , Corantes Fluorescentes/química , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/metabolismo , Antracenos/química , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Humanos , Masculino , Modelos Químicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Neoplasias da Próstata/genética , Espectrofotometria Ultravioleta , Temperatura , Termodinâmica
5.
Mol Cancer ; 9: 241, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20843307

RESUMO

BACKGROUND: Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. RESULTS: Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. CONCLUSIONS: Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , RNA Polimerase III/metabolismo , RNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Fator 2 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Antígenos Nucleares do Vírus Epstein-Barr/genética , Imunofluorescência , Humanos , Immunoblotting , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase III/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo
6.
Mol Cancer ; 9: 1, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20051109

RESUMO

BACKGROUND: The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB. RESULTS: In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied. CONCLUSIONS: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação para Baixo/genética , Antígenos Nucleares do Vírus Epstein-Barr/química , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Virais/genética , Herpesvirus Humano 4/genética , Humanos , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Deleção de Sequência , Fator de Transcrição RelA/metabolismo , Ativação Transcricional/genética , Regulação para Cima/genética
7.
Pathogens ; 9(7)2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32708289

RESUMO

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFß) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP "signature" in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.

8.
Eur J Med Genet ; 61(4): 230-234, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29225144

RESUMO

The genetic association between autism spectrum disorder (ASD) and psychotic disorders such as schizophrenia is complicated and mirrors the clinical overlap between these conditions to some degree. However, no studies to date have examined the genetics of individuals dually diagnosed with both ASD and psychosis. In this study, we present findings of copy number variants (CNVs) from a study of 116 well-characterised individuals with this dual diagnosis. DNA was extracted and arrayed using the Affymetrix CytoScan HD 2.8M array or the Affymetrix Cytogenetics arrays and compared with existing samples from the Database of Genomic Variants and the Simons Simplex Collection of CNVs from individuals with ASD and their families. Twenty-seven novel CNVs ≥20k base pairs were identified in the sample, most occurring in only a single individual, although two were found in two female participants. Forty-nine rare CNVs (<1.5% rate in general population) were also found at significantly higher frequencies than expected. The findings may provide evidence for areas of further study in the understanding of the development of both ASD and psychosis due to the number of affected genetic regions that have not previously been linked to these conditions.


Assuntos
Transtorno do Espectro Autista/genética , Variações do Número de Cópias de DNA , Transtornos Psicóticos/genética , Transtorno do Espectro Autista/complicações , Humanos , Transtornos Psicóticos/complicações
9.
PLoS One ; 7(7): e41055, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815911

RESUMO

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.


Assuntos
Carcinoma/metabolismo , Carcinoma/virologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Homozigoto , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição Gênica , Regulação para Cima , Proteínas da Matriz Viral/biossíntese , Proteínas Virais/biossíntese
10.
J Gen Virol ; 89(Pt 11): 2806-2820, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18931079

RESUMO

SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell-cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1beta and IL-1alpha was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.


Assuntos
Herpesvirus Humano 4/patogenicidade , Queratinócitos/fisiologia , Proteínas da Matriz Viral/efeitos adversos , Anticorpos Antivirais , Diferenciação Celular , Divisão Celular/genética , Linhagem Celular , Movimento Celular , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Queratinócitos/patologia , Hibridização de Ácido Nucleico , RNA Viral/genética , RNA Viral/isolamento & purificação , Ferimentos e Lesões/virologia
11.
J Gen Virol ; 89(Pt 11): 2833-2842, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18931081

RESUMO

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/irrigação sanguínea , Neoplasias Nasofaríngeas/genética , Neovascularização Patológica/genética , Fator de Transcrição AP-1/fisiologia , Adenocarcinoma , Linhagem Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genoma Viral , Humanos , Imuno-Histoquímica , Interleucina-8/fisiologia , Rim/embriologia , Microtúbulos/patologia , Neoplasias Nasofaríngeas/virologia , Neovascularização Patológica/virologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
12.
J Immunol ; 179(5): 2705-12, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17709483

RESUMO

Activated B cells reacting to small amounts of CD40L (CD154) maintain homeostasis by suppressing default apoptosis. Additional outcomes, particularly differentiation, demand higher CD40 occupancy. Here, focusing on survival, we compared changes in the transcriptome of pleiotropically competent, early passage L3055 Burkitt's lymphoma cells confronted with low (picomolar) and high (nanomolar) concentrations of CD154 to gain insight into how a single receptor sets these distinct phenotypes. Of 267 genes altering transcriptional activity in response to strong CD154 tone, only 25 changed coordinately on low receptor occupancy. Seven of the top nine common up-regulated genes were targets of NF-kappaB. Direct measurement and functional inhibition of the NF-kappaB pathway revealed it to be central to a CD40-dependent survival signature. Although the canonical NF-kappaB axis was engaged by both signaling strengths equally, robust alternative pathway activation was a feature selective to a strong CD40 signal. Discriminatory exploitation of the two separate arms of NF-kappaB activation may indicate a principle whereby a cell senses and reacts differentially to shifting ligand availability. Identifying components selectively coupling CD40 to each axis could indicate targets for disruption in B cell pathologies underpinned by ectopic and/or hyper-CD154 activity such as neoplasia and some autoimmunities.


Assuntos
Linfoma de Burkitt/genética , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Ligante de CD40/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , NF-kappa B/análise , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcrição Gênica
13.
J Biol Chem ; 281(45): 33871-80, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16956891

RESUMO

The EBER genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These EBER transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of EBER RNA. These include phosphorylation of ATF2, which interacts with EBER promoters. A second is induction of TFIIIC, a pol III-specific factor that activates EBER genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate EBER expression in vivo, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/fisiologia , RNA Polimerase III/metabolismo , RNA Viral/genética , Fatores de Transcrição TFIII/metabolismo , Transcrição Gênica , Western Blotting , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutagênese Sítio-Dirigida , Proteínas Associadas à Matriz Nuclear , Fatores de Transcrição de Octâmero , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , Transfecção
14.
Virology ; 304(2): 342-51, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12504574

RESUMO

Monocytes and macrophages play a central role in viral infections, as a target for viruses and in activation of both innate and adaptive immune responses. Epstein-Barr virus (EBV) has evolved elaborate strategies to dampen the immune system and to persist within the host. There is evidence that the product of the BCRF-1 open reading frame of EBV, viral interleukin-10 (vIL-10), inhibits the capacity of monocytes/macrophages to induce T cell activation, but the full mechanism of this effect is unknown. To determine whether this effect might involve modulation of the expression of accessory molecules known to be important in T cell activation, we analyzed by flow cytometry the influence of vIL-10 on the basal as well as on IFN-gamma-induced up-regulation of HLA molecules, ICAM-1, and two members of the B7 family B7.1 (CD80) and B7.2 (CD86) at the surface of human monocytes. Viral IL-10 in a concentration-dependent manner inhibited both basal- and IFN-gamma-induced HLA-class II, ICAM-1 (basal levels of ICAM-2 and ICAM-3 is unaffected), CD80, and CD86 up-regulation when added simultaneously with IFN-gamma. In contrast, complete inhibition of HLA-class I expression on monocytes/macrophages occurred only when vIL-10 was present 2 h prior to the addition of IFN-gamma, implying that vIL-10 affects an early step in the IFN-gamma signaling pathway. As both monocytes and macrophages can be infected by EBV, we propose that vIL-10-mediated impairment of monocyte/macrophage antigen-presenting function could help the virus-infected cells to avoid detection by the host's T cells on one hand and contribute to its immunosuppressive properties on the other.


Assuntos
Antígeno B7-1/análise , Herpesvirus Humano 4/patogenicidade , Antígenos de Histocompatibilidade Classe I/análise , Molécula 1 de Adesão Intercelular/análise , Interleucina-10/toxicidade , Monócitos/efeitos dos fármacos , Proteínas Virais/toxicidade , Apresentação de Antígeno/efeitos dos fármacos , Antígenos CD/análise , Antígeno B7-2 , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Interferon gama/farmacologia , Macrófagos/química , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/análise , Monócitos/química
15.
J Immunol ; 173(1): 321-31, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15210790

RESUMO

The role of monocytes and macrophages during EBV infection is not clear. The interaction of EBV with human monocytes was investigated in terms of cell survival and morphological and phenotypic changes to gain a better understanding of the role of these cells during EBV infection. We show that EBV infection of PBMCs rescues monocytes from undergoing spontaneous apoptosis and dramatically enhances their survival. Results obtained with heat-inactivated virus, neutralizing anti-EBV mAb 72A1 and recombinant gp350, suggest that enhancement of viability by EBV requires both infectious virus and interaction between gp350 and its receptor. IFN-alpha either secreted within 24 h from PBMCs upon infection with EBV or exogenously added to unstimulated monocytes inhibited spontaneous apoptosis, indicating that induction of IFN-alpha is an early important survival signal responsible for the delay in the apoptosis of monocytes. EBV infection also induced acute maturation of monocytes to macrophages with morphological and phenotypic characteristics of potent APCs. Monocytes exposed to EBV became larger in size with increased granularity and expressed considerably higher levels of membrane HLA classes I and II, ICAM-1, CD80, CD86, and CD40 compared with uninfected cultures. These observations provide the first immunoregulatory links among EBV, IFN-alpha, and monocyte survival and maturation and importantly raise the possibility that these cells may serve as a vehicle for the dissemination of the virus as well as being active participants in eliciting anti-EBV T cell responses during acute infection.


Assuntos
Herpesvirus Humano 4/fisiologia , Interferon-alfa/biossíntese , Monócitos/fisiologia , Apoptose , Sobrevivência Celular , Humanos , Proteínas da Matriz Viral/fisiologia
16.
Ai Zheng ; 22(5): 481-5, 2003 May.
Artigo em Zh | MEDLINE | ID: mdl-12753707

RESUMO

BACKGROUND & OBJECTIVE: It has been proved that Epstein- Barr virus (EBV) latent membrane protein 1 (EBV-LMP1) can induce the expression of matrix metalloproteinase-9 (MMP-9). This study was designed to investigate the effect of EBV-LMP1 on related factors of metastasis of nasopharyngeal carcinoma cell line CNE1. METHODS: Expression of MMP-9 was studied in human NPC cell lines cultured in vitro: CNE1 (well differentiated cell line of NPC) and CNE1-GL (CNE1 cell line transfected with an eukaryotic LMP1-expression plasmid) by SP immunohistochemistry and Western blot analysis. Cell-matrix adhesion assay was used to study the adhesive ability of CNE1-GL cells. The effects of LMP1 on the invasion and migration of CNE1 cells were investigated by transwell methods. RESULTS: MMP-9 was expressed in both cell lines but the intensity of the staining was different. The positive rates of expression of MMP-9 in CNE1 and CNE1-GL cells were 30.2% and 98.2%, respectively (P< 0.05). The increased expression of MMP-9 was also shown in CNE1-GL cells by Western blot analysis. Cell-matrix adhesion assay showed that the adhesive ability of CNE1-GL with the matrix (mean A value: 1.2508+/-0.0711) was higher than that of CNE1 cell (mean A value: 0.9519+/-0.068) (P< 0.001). Invasion assay and migration assay showed that the invasion and migration of CNE1-GL cell were higher than those of CNE1 cells (P< 0.01). CONCLUSION: The transfection of LMP1 can increase the expression of MMP-9 in CNE1 cells. Abilities of adhesion, migration, and invasion of CNE1 cell were induced by LMP1. It is suggested that MMP-9 may have a role in the LMP1-induced acceleration of invasion and metastasis of NPC cells.


Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias Nasofaríngeas/patologia , Proteínas da Matriz Viral/farmacologia , Proteínas Virais/farmacologia , Adesão Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Metástase Neoplásica , Células Tumorais Cultivadas
17.
J Gen Virol ; 83(Pt 7): 1621-1633, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12075080

RESUMO

A gammaherpesvirus related to Epstein-Barr virus (EBV; Human herpesvirus 4) infects otherwise healthy common marmosets (Callithrix jacchus). Long-term culture of common marmoset peripheral blood lymphocytes resulted in outgrowth of spontaneously immortalized lymphoblastoid cell lines, primarily of B cell lineage. Electron microscopy of cells and supernatants showed herpesvirus particles. There were high rates of serological cross-reactivity to other herpesviruses (68-86%), but with very low geometric mean antibody titres [1:12 to human herpesvirus 6 and 1:14 to Herpesvirus papio (Cercopithecine herpesvirus 12)]. Sequence analysis of the conserved herpesvirus DNA polymerase gene showed that the virus is a member of the lymphocryptovirus subgroup and is most closely related to a lymphocryptovirus from rhesus macaques and is closely related to EBV and Herpesvirus papio. High seroprevalence (79%, with geometric mean antibody titre of 1:110) among 28 common marmosets from two geographically distinct colonies indicated that the virus is likely present in many common marmosets in captivity. A New World primate harbouring a lymphocryptovirus suggests that this subgroup arose much earlier than previously thought.


Assuntos
Callithrix/virologia , Gammaherpesvirinae/classificação , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Callithrix/imunologia , Linhagem Celular , Reações Cruzadas , DNA Polimerase Dirigida por DNA/genética , Gammaherpesvirinae/genética , Gammaherpesvirinae/imunologia , Leucócitos Mononucleares/ultraestrutura , Leucócitos Mononucleares/virologia , Microscopia Eletrônica , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Estudos Soroepidemiológicos
18.
Cytokine ; 17(1): 1-13, 2002 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-11886166

RESUMO

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Endotoxinas/química , Proteínas de Escherichia coli , Herpesvirus Humano 4/metabolismo , Interleucina-10/biossíntese , Interleucina-10/química , Proteínas de Transporte de Monossacarídeos , Amilose/química , Animais , Proteínas de Transporte/química , Cromatografia , Clonagem Molecular , DNA Complementar/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Heparina/metabolismo , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Ligantes de Maltose , Octoxinol , Fases de Leitura Aberta , Polietilenoglicóis/farmacologia , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química , Sefarose/metabolismo
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