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1.
Clin Dermatol ; 38(6): 702-711, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33341203

RESUMO

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a severe cutaneous drug reaction characterized by fever, lymphadenopathy, hematologic abnormalities, multisystem involvement, and viral reactivation. Although most patients with DRESS syndrome are able to fully recover, a subset of patients go on to have a prolonged course with recurrence, and/or autoimmune complications. Severe systemic involvement is associated with significant morbidity and mortality. Viral reactivation, especially of human herpes virus 6, Epstein-Barr virus, and cytomegalovirus, is a common feature of DRESS, with a high viral load and antibody titers being associated with poor outcomes. Aside from prompt discontinuation of the offending drug, treatment for patients with significant disease consists of systemic therapy with corticosteroids.


Assuntos
Síndrome de Hipersensibilidade a Medicamentos/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Eosinofilia/induzido quimicamente , Preparações Farmacêuticas , Corticosteroides/uso terapêutico , Citomegalovirus , Síndrome de Hipersensibilidade a Medicamentos/terapia , Síndrome de Hipersensibilidade a Medicamentos/virologia , Feminino , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 6 , Humanos , Masculino , Prognóstico , Índice de Gravidade de Doença , Carga Viral , Ativação Viral
2.
Anticancer Res ; 40(11): 5951-5968, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109533

RESUMO

BACKGROUND/AIM: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. A systematic review to evaluate the association between Epstein-Barr Virus (EBV) and programmed death ligand-1 (PD-L1) in DLBCL biopsy was conducted. MATERIALS AND METHODS: Only studies comparing EBV+ and EBV- groups were eligible following database search. Prevalence ratios were calculated for results comparison. The EBV impact on PD-L1 positivity in tumour cells and its microenvironment was analysed. RESULTS: With 270 records screened, eleven cross-sectional studies were identified for final review. Eight studies investigated PD-L1 expression in tumour cells and found an EBV trend unlikely, while four studies found an increase in its expression in the tumour microenvironment. Nine studies showed that EBV+ cases were more commonly of non-germinal centre B-cell origin. Four studies examined genetic aberrations, but no definite consensus was reached. CONCLUSION: A non-EBV related mechanism is likely related to increased PD-L1 expression, with relevance to the cell of origin.


Assuntos
Antígeno B7-H1/metabolismo , Herpesvirus Humano 4/fisiologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/virologia , Idoso , Feminino , Humanos , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Viés de Publicação , Microambiente Tumoral
3.
PLoS One ; 15(8): e0238062, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841308

RESUMO

This retrospective multicenter cohort study investigated the kinetics (ascending and descending phases) of cytomegalovirus (CMV) and Epstein-Barr virus (EBV)-DNA in whole blood (WB) and plasma samples collected from adult kidney transplant (KT) recipients. CMV-DNA kinetics according to antiviral therapy were investigated. Three hundred twenty-eight paired samples from 42 episodes of CMV infection and 157 paired samples from 26 episodes of EBV infection were analyzed by a single commercial molecular method approved by regulatory agencies for both matrices. CMV-DNAemia followed different kinetics in WB and plasma. In the descending phase of infection, a slower decay of viral load and a higher percentage of CMV-DNA positive samples were observed in plasma versus WB. In the 72.4% of patients receiving antiviral therapy, monitoring with plasma CMV-DNAemia versus WB CMV-DNAemia could delay treatment interruption by 7-14 days. Discontinuation of therapy based on WB monitoring did not result in relapsed infection in any patients. Highly different EBV-DNA kinetics in WB and plasma were observed due to lower positivity in plasma; EBV positive samples with a quantitative result in both blood compartments were observed in only 11.5% of cases. Our results emphasize the potential role of WB as specimen type for post-KT surveillance of both infections for disease prevention and management.


Assuntos
Citomegalovirus/genética , DNA Viral/sangue , Herpesvirus Humano 4/genética , Transplante de Rim , Adulto , Antivirais/farmacologia , Estudos de Coortes , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/fisiologia , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/fisiologia , Humanos , Imunossupressores/farmacologia , Cinética , Estudos Retrospectivos
4.
PLoS Pathog ; 16(6): e1008590, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542010

RESUMO

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.


Assuntos
Infecções por Vírus Epstein-Barr , Antígenos Nucleares do Vírus Epstein-Barr/genética , Deleção de Genes , Herpesvirus Humano 4/fisiologia , Doença de Hodgkin , Linfoma Difuso de Grandes Células B , Proteínas Virais/genética , Latência Viral , Animais , Linhagem Celular , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/patogenicidade , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/virologia , Camundongos , Proteínas Virais/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(22): 12368-12374, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409608

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes life-long infection and increases the risk for the development of several cancers and autoimmune diseases. The mechanisms by which chronic EBV infection leads to subsequent disease remain incompletely understood. Lytic reactivation plays a central role in the development of EBV-driven cancers and may contribute to other EBV-associated diseases. Thus, the clinical use of antivirals as suppressive therapy for EBV lytic reactivation may aid efforts aimed at disease prevention. Current antivirals for EBV have shown limited clinical utility due to low potency or high toxicity, leaving open the need for potent antivirals suitable for long-term prophylaxis. In the present study, we show that tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), drugs with excellent safety profiles used clinically for HIV prevention, inhibit EBV lytic DNA replication, with respective IC50 values of 0.30 µM and 84 nM. In a cell-based assay, TAF was 35- and 24-fold and TDF was 10- and 7-fold more potent than acyclovir and penciclovir, respectively, and TAF was also twice as potent as ganciclovir. The active metabolite of tenofovir prodrugs, tenofovir-diphosphate, inhibited the incorporation of dATP into a primed DNA template by the EBV DNA polymerase in vitro. In contrast to acyclovir, treatment of cells during latency for 24 h with TAF still inhibited EBV lytic DNA replication at 72 h after drug was removed. Our results suggest that tenofovir prodrugs may be particularly effective as inhibitors of EBV lytic reactivation, and that clinical studies to address critical questions about disease prevention are warranted.


Assuntos
Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Tenofovir/farmacologia , Proteínas Virais/antagonistas & inibidores , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Pró-Fármacos/farmacologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Cancer Sci ; 111(7): 2598-2607, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32338409

RESUMO

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein-Barr virus (EBV)-associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV-infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV-infected TMK1 cells and these cell populations showed high spheroid-forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9-/- cells. Sphere-forming ability was dependent on the nuclear factor-κB (NF-κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11-7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere-forming ability of TMK1 cells, which was mediated through NF-κB signaling. Together, these results indicate that CD44v6v9+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF-κB pathway. Future studies should investigate CD44v6/v9+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/etiologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunofenotipagem , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Cell ; 78(4): 653-669.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32315601

RESUMO

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.


Assuntos
Linfoma de Burkitt/patologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Viral , Latência Viral , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/virologia , Proliferação de Células , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS Pathog ; 16(3): e1008447, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176739

RESUMO

Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/fisiologia , Motivos de Aminoácidos , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr/genética , Humanos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo
9.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32213613

RESUMO

Interferon alpha (IFN-α) and IFN-ß are type I IFNs that are induced by virus infection and are important in the host's innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation.IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host's antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Interferon Tipo I/imunologia , Transdução de Sinais/imunologia , Proteínas Virais de Fusão/imunologia , Ativação Viral/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Células HEK293 , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Interferon gama/genética , Interferon gama/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , TYK2 Quinase/genética , TYK2 Quinase/imunologia , Proteínas Virais de Fusão/genética , Ativação Viral/genética
10.
Asian Pac J Cancer Prev ; 21(3): 693-698, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32212795

RESUMO

BACKGROUND: Epstein-Barr virus (EBV) is associated with different malignant diseases, such as Hodgkin lymphoma (HL) and lymphoproliferative disorders. Patients with hematologic malignancies by variable severity could be suspected for the infection with different types of this virus. This preliminary study reported the genotyping and related viral load of Epstein-Barr virus in Iranian patients with hematologic malignancies for estimation of possible factors affecting malignancy. METHODS: Peripheral blood mononuclear cells (PBMC) of HL (n=20), NHL (n=29), acute lymphocytic leukemia (ALL) (n=18) and chronic lymphocytic leukemia (CLL) (n=12) were obtained. After DNA extraction, a nested-PCR and a conventional-PCR targeting EBNA-2 and EBNA-3C genes were performed. A real-time PCR assay for viral load quantitation carried out. Standard curve analysis used for evaluation of amplification specificity. RESULTS: Of 79 included patients, 34 (43%) were EBV positive. There were 23.5% (8/34), 38.2% (13/34), 23.5% (8/34), 14.8% (5/34) in HL, NHL, ALL and CLL groups, respectively. Also, the main genotype was genotype I (91.2%) which it follows by 8.8% (3/34) genotype II. The real-time PCR assay showed the mean viral load ± std. deviation was 2.75×105 ± 1.202×106 copies/µg DNA and the higher viral load was seen in NHL patients. CONCLUSION: This preliminary investigation in Iran shows that the main EBV genotype into our region probably is genotype I (91.2%) which it is similar to others. We could not find any statistically significant association between the virus infection and viral load with any specific disease and patients' demographic data. 
.


Assuntos
Infecções por Vírus Epstein-Barr/epidemiologia , Neoplasias Hematológicas/virologia , Herpesvirus Humano 4/genética , Adulto , Idoso , DNA Viral/genética , Infecções por Vírus Epstein-Barr/complicações , Feminino , Genótipo , Herpesvirus Humano 4/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Carga Viral
11.
Cancer Sci ; 111(5): 1818-1828, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32119176

RESUMO

Epstein-Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing (-seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3- enhancers in EBV-infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein-Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation-seq analysis on ATF3 binding sites and RNA-seq analysis on ATF3 knocked-down MKN7_EB revealed 96 genes targeted by ATF3-activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV-positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV-infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV-positive GC.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Elementos Facilitadores Genéticos , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/fisiologia , Neoplasias Gástricas/genética , Fator 3 Ativador da Transcrição/genética , Apoptose/genética , Sítios de Ligação , Linhagem Celular , Proliferação de Células/genética , Epigenoma , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , Mutação , Transcriptoma
12.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132242

RESUMO

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Sangue Fetal/metabolismo , Herpesvirus Humano 4/genética , Linfoma/virologia , Animais , Linfócitos B/virologia , Transformação Celular Viral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Células Matadoras Naturais/imunologia , Linfoma/genética , Linfoma/patologia , Linfoma de Células B , Camundongos , Mutagênese Sítio-Dirigida , Análise de Sequência de RNA , Deleção de Sequência , Linfócitos T/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral/genética
13.
PLoS One ; 15(2): e0228451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32017805

RESUMO

Viral reactivation occurs frequently in the context of immunodeficiency and immunosuppression after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and can cause severe complications. The aim of this single-center retrospective analysis was to characterize viral infections in the first year after HSCT, to investigate risk factors and to study the impact of viral infections on transplantation outcome. This will facilitate the identification of at-risk patients and the development of new preventive strategies. 107 pediatric allo-HSCT from January 2005 through December 2015 were analyzed for infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), adenovirus (ADV), herpes simplex virus (HSV) and varicella zoster virus (VZV). Viral infections were detected after 68.2% of transplantations. The viruses most commonly encountered were HHV-6 (36/107) and EBV (30/107). Severe viral disease was rare (7/107) and none of the patients died as result of viral reactivation. Important risk factors for viral infections were higher age at HSCT, donor type and occurrence of acute graft-versus-host disease (aGvHD). Especially for EBV, transplant from an unrelated donor and in-vivo T-cell depletion (TCD) had a significant effect on infection rates, whereas for CMV the strongest effect was seen by donor and recipient serostatus with recipient seropositivity most predictive for reactivation. The occurrence of severe aGvHD was associated with EBV and ADV infections. For HSV, the recipient serostatus was identified as prognostic factor for HSV infections, while we found higher age at time of HSCT as risk factor for VZV infections. The overall survival of patients with or without viral infections did not differ significantly. Interestingly, when looking at the 85 patients in our cohort who had received an HSCT for a malignant disease, a tendency towards lower relapse rates was seen in patients affected by viral infections (HR 0.51, 95% CI 0.25 - 1.06, p = 0.072). Viral reactivations are common after pediatric allo-HSCT, though severe complications were rare in our collective. Determining risk factors for viral reactivations may help to identify patients in need of intensified monitoring and to individualize preventive strategies.


Assuntos
Doença Enxerto-Hospedeiro/epidemiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Viral , Viroses/epidemiologia , Adenoviridae/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Citomegalovirus/fisiologia , Feminino , Doença Enxerto-Hospedeiro/virologia , Herpesvirus Humano 3/fisiologia , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 6/fisiologia , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Medição de Risco , Simplexvirus/fisiologia , Análise de Sobrevida , Transplante Homólogo/efeitos adversos , Viroses/virologia , Adulto Jovem
14.
Nat Commun ; 11(1): 685, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019925

RESUMO

IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFß-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin-1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer.


Assuntos
Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Quinase I-kappa B/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Herpesvirus Humano 4/genética , Humanos , Quinase I-kappa B/genética , Linfoma/enzimologia , Linfoma/genética , Linfoma/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais
16.
Nat Commun ; 11(1): 877, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054837

RESUMO

Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.


Assuntos
Sítios de Ligação Microbiológicos/genética , Sítios de Ligação Microbiológicos/fisiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/virologia , Epigênese Genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Plasmídeos/genética , Latência Viral/genética , Latência Viral/fisiologia
17.
Virology ; 540: 160-164, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31928997

RESUMO

The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 - and - expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.


Assuntos
Apoptose , Linfócitos B/metabolismo , Linfócitos B/virologia , Caspase 9/metabolismo , Transformação Celular Viral , Fator de Transcrição STAT3/metabolismo , Linfócitos B/patologia , Caspase 7/metabolismo , Caspase 9/genética , Herpesvirus Humano 4/fisiologia , Humanos , Modelos Biológicos , Fosforilação , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética
18.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941781

RESUMO

The binding of Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) to the latent replication origin (oriP) triggers multiple downstream events to support virus-induced pathogenesis and tumorigenesis. Although EBV is widely recognized as a B-lymphotropic infectious agent, little is known about how tissue-specific factors are involved in the establishment of latency. Here, we showed that EBNA1 binds B cell activator PAX5 to promote EBNA1/oriP-dependent binding and transcription. In addition to showing that short hairpin RNA (shRNA)-mediated PAX5 knockdown substantially abrogated the above EBNA1-dependent functions, two mini-EBV reporter plasmids were used to perform nonlytic nano-luciferase (nLuc) activity and chromatin immunoprecipitation (ChIP) assays to show how EBNA1 cooperates with PAX5 to activate the transcription at the oriP site. The expression plasmids of two PAX5 mutants, V26G (EBNA1 binding mutant) and P80R (which remained EBNA1 associated), were used to assess their capability to restore the defects caused by PAX5 depletion in EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the mini-EBV episome reporter in BJAB cells stably expressing EBNA1 or that of the EBV genome in EBV-infected BJAB cells. Since p300 is known to be associated with PAX5, we showed that the loss of function of the P80R mutant in support of EBNA1/oriP-mediated transcription under PAX5 depletion conditions was linked to its defective binding to p300. ChIP-quantitative PCR (qPCR) confirmed that P80R indeed failed to recruit p300 to the oriP DNA. Our discovery suggests that EBV has evolved an exquisite strategy to take advantage of tissue-specific factors to enable the establishment of viral latency.IMPORTANCE Although B cells are known to be the primary target for EBV infection, there is limited knowledge regarding the mechanism that determines this preferable tissue tropism. An in-depth understanding of the potential link of tissue-specific factors with the viral genes and their functioning is key to deciphering how EBV induces persistent infection in the distinct types of host cells. In this study, a substantial protein-protein interaction mediated by the B cell-specific activator PAX5 and EBNA1 was identified as the general requirement for the binding of EBNA1 to the latent replication origin and for downstream events. Of importance, the EBNA1-PAX5-p300 network is directly linked to EBNA1-dependent transcription. These findings suggest that targeting the viral gene-associated tissue-specific factors may lead to new therapeutic strategies for EBV-associated malignancies.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Fator de Transcrição PAX5/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Mutação , Plasmídeos , Ligação Proteica , Mapas de Interação de Proteínas , RNA Interferente Pequeno , Origem de Replicação , Replicação Viral
19.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941784

RESUMO

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle.IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular "prolytic" state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.


Assuntos
Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/genética , Transativadores/metabolismo , Transcriptoma , Latência Viral , Apoptose , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Inflamação , Fenótipo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Ativação Transcricional , Carga Viral
20.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941785

RESUMO

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/química , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Virais/química , Linhagem Celular Tumoral , Nucléolo Celular/química , Núcleo Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Genes myc , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Leucócitos Mononucleares , Microscopia de Fluorescência , Prolina/química , Regiões Promotoras Genéticas , Domínios Proteicos
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