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Abstract
Abstract
Background: There are few reports about the interactions of EBV with peripheral T-cells, especially during the early phase of infection. Objective: Demonstrate the capability of EBV to infect and replicate in human peripheral T-cells in vitro. Methods: After treating with EBV, the susceptibility of in vitro EBV infection into T-cells was confirmed using electron microscopy, the expression of EBV mRNA using RT-PCR, and the expression of EBV proteins using Western blot analysis. The expression of CD19 and CD21 mRNA was determined using RT-PCR. The induction of cell death was measured using trypan blue exclusion assay. Results: The susceptibility of in vitro EBV infection was confirmed by the presence of virus particles in the cytoplasm. The entering to lytic infection was confirmed by detection the expression of EBV lytic (BZLF1) mRNA, and the expression of late lytic proteins (VCA and gp350/220). The expression of CD19 and CD21 were not observed using RT-PCR. The interactions of EBV with T-cells leaded to induction of T-cell death. Conclusion: Peripheral T-cells are a direct target of EBV infection. At the beginning of infection by EBV, EBV infection of T-cells leads to the entering into lytic virus replication. EBV binds to these cells through a receptor distinct from the CD21.
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Analysis of an ankyrin-like region in Epstein Barr Virus encoded (EBV) BZLF-1 (ZEBRA) protein: implications for interactions with NF-κB and p53. Virol J 2011; 8:422. [PMID: 21892957 PMCID: PMC3180424 DOI: 10.1186/1743-422x-8-422] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2011] [Accepted: 09/05/2011] [Indexed: 12/19/2022] Open
Abstract
Background The carboxyl terminal of Epstein-Barr virus (EBV) ZEBRA protein (also termed BZLF-1 encoded replication protein Zta or ZEBRA) binds to both NF-κB and p53. The authors have previously suggested that this interaction results from an ankyrin-like region of the ZEBRA protein since ankyrin proteins such as IκB interact with NF-κB and p53 proteins. These interactions may play a role in immunopathology and viral carcinogenesis in B lymphocytes as well as other cell types transiently infected by EBV such as T lymphocytes, macrophages and epithelial cells. Methods Randomization of the ZEBRA terminal amino acid sequence followed by statistical analysis suggest that the ZEBRA carboxyl terminus is most closely related to ankyrins of the invertebrate cactus IκB-like protein. This observation is consistent with an ancient origin of ZEBRA resulting from a recombination event between an ankyrin regulatory protein and a fos/jun DNA binding factor. In silico modeling of the partially solved ZEBRA carboxyl terminus structure using PyMOL software demonstrate that the carboxyl terminus region of ZEBRA can form a polymorphic structure termed ZANK (ZEBRA ANKyrin-like region) similar to two adjacent IκB ankyrin domains. Conclusions Viral capture of an ankyrin-like domain provides a mechanism for ZEBRA binding to proteins in the NF-κB and p53 transcription factor families, and also provides support for a process termed "Ping-Pong Evolution" in which DNA viruses such as EBV are formed by exchange of information with the host genome. An amino acid polymorphism in the ZANK region is identified in ZEBRA from tumor cell lines including Akata that could alter binding of Akata ZEBRA to the p53 tumor suppressor and other ankyrin binding protein, and a novel model of antagonistic binding interactions between ZANK and the DNA binding regions of ZEBRA is suggested that may be explored in further biochemical and molecular biological models of viral replication.
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Dittmar T, Zänker KS. Horizontal gene transfers with or without cell fusions in all categories of the living matter. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2011; 714:5-89. [PMID: 21506007 PMCID: PMC7120942 DOI: 10.1007/978-94-007-0782-5_2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. "Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor" remain cellular life's basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.
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Affiliation(s)
- Thomas Dittmar
- Inst. Immunologie, Universität Witten/Herdecke, Stockumer Str. 10, Witten, 58448 Germany
| | - Kurt S. Zänker
- Institute of Immunologie, University of Witten/Herdecke, Stockumer Str. 10, Witten, 58448 Germany
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4
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Abstract
Abstract
Background: Although the presence of Epstein-Barr virus (EBV) in different T-cell malignancies has been widely reported, there is very few data available for EBV infection of normal T cells. This leads to the lack of knowledge on the early events after T cell infection. Objective: Investigate the early events occurring after normal human peripheral T-cells are infected with EBV in vitro. Methods: T-cells were treated with EBV in vitro. The expression of tumor necrosis factor- α (TNF-α) mRNA were determined using reverse-transcription (RT)-PCR, and the level of TNF-α and interferon- γ (IFN-γ) in the culture supernatant were measured using ELISA. The effect of virus inactivation on cytokine induction from T-cells was also determined. Results: At the beginning of T cell infection by EBV, the expression of several lytic EBV transcripts (BALF5, BcLF1, and BLLF1) were observed using RT-PCR. This indicated the susceptibility of in vitro EBV infection and the entering lytic cycle of EBV-infected T-cells. The interactions of EBV with T-cells lead to induction of inflammatory cytokines, tumour necrosis factor- α (TNF-α) and interferon- γ (IFN-γ), production from the T-cells. Inactivation of the virus by UV irradiation eliminated the TNF-α and IFN-γ induction by EBV, suggesting the involvement in the expression of viral gene(s). Conclusion: This in vitro analysis demonstrated the cytokine induction by EBV after primary infection of T-cells.
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Katsumura KR, Maruo S, Wu Y, Kanda T, Takada K. Quantitative evaluation of the role of Epstein-Barr virus immediate-early protein BZLF1 in B-cell transformation. J Gen Virol 2009; 90:2331-2341. [PMID: 19553389 DOI: 10.1099/vir.0.012831-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
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.
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Affiliation(s)
- Koichi Ricardo Katsumura
- Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
| | - Seiji Maruo
- Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
| | - Yi Wu
- Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
| | - Teru Kanda
- Research Center for Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
| | - Kenzo Takada
- Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
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6
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Dreyfus DH. Role of T Cells in EBV-Infected Systemic Lupus Erythematosus Patients. THE JOURNAL OF IMMUNOLOGY 2005; 175:3460; author reply 3461. [PMID: 16148086 DOI: 10.4049/jimmunol.175.6.3460] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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Dreyfus DH. Immunopathology associated with Epstein-Barr virus (EBV) infection: Evidence for interactions with T-lymphocyte EBV receptor CD21. ACTA ACUST UNITED AC 2005. [DOI: 10.1016/j.cair.2005.01.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Wang Y, Luo B, Yan LP, Huang BH, Zhao P. Relationship between Epstein-Barr virus-encoded proteins with cell proliferation, apoptosis, and apoptosis-related proteins in gastric carcinoma. World J Gastroenterol 2005; 11:3234-9. [PMID: 15929173 PMCID: PMC4316054 DOI: 10.3748/wjg.v11.i21.3234] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the interrelationship between Epstein-Barr virus (EBV)-encoded proteins and cell proliferation, apoptosis and apoptosis-related proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis.
METHODS: Tissues from 13 cases of EBV-associated gastric carcinoma (EBVaGC) and 45 cases of matched EBV-negative gastric carcinoma (EBVnGC) were collected, and then subjected to analysis for apoptotic index (AI) using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Nuclear cell proliferation-associated antigen ki-67 index (KI), bcl-2, and p53 expression were examined by immunohistochemistry. p53 mutation in exons 5-8 of 13 EBVaGC cases was determined by single-strand conformation polymorphism (SSCP) and DNA sequencing. RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (LMP) 1, immediately early gene BZLF1 and early genes BARF1 and BHRF1 in 13 EBVaGC cases.
RESULTS: The percentage of AI, KI and p53 overexpression was significantly lower in the EBVaGC group than in the EBVnGC group. However, bcl-2 expression did not show significant difference between the two groups. p53 gene mutations were not found in 13 EBVaGCs. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the thirteen cases exhibited BZLF1 transcripts and two exhibited BHRF1 transcripts. BARF1 mRNA was detected in six cases.
CONCLUSION: Lower AI and KI may reflect a low biological activity in EBVaGC. EBV infection is associated with p53 abnormal expression but not bcl-2 protein in EBVaGC. BZLF1, BARF1, and BHRF1 may play important roles in inhibiting cell apoptosis and tumorigenesis of EBVaGC through different pathways.
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Affiliation(s)
- Yun Wang
- Department of Microbiology, Qingdao University Medical College, Number 38 of Dengzhou Road, Qingdao 266021, Shandong Province, China
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Luo B, Wang Y, Wang XF, Liang H, Yan LP, Huang BH, Zhao P. Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas. World J Gastroenterol 2005; 11:629-33. [PMID: 15655811 PMCID: PMC4250728 DOI: 10.3748/wjg.v11.i5.629] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level.
METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs.
RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (χ2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLF1 transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected.
CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.
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Affiliation(s)
- Bing Luo
- Department of Microbiology, Qingdao University Medical College, Number 38 of Dengzhou Road, Qingdao 266021, Shandong Province, China.
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Tardif M, Savard M, Flamand L, Gosselin J. Impaired protein kinase C activation/translocation in Epstein-Barr virus-infected monocytes. J Biol Chem 2002; 277:24148-54. [PMID: 11971896 DOI: 10.1074/jbc.m109036200] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Infection of human monocytes by Epstein-Barr virus (EBV) has been linked to a decrease in the production of proinflammatory mediators as well as an impairment of phagocytosis. Considering the key role of protein kinases C (PKCs) in many biological functions of monocytes, including phagocytosis, we investigated the effects of EBV on the PKC activity in infected monocytes. Our results indicate that infection of monocytes by EBV impairs both phorbol 12-myristate 13-acetate (PMA)-induced translocation of PKC isozymes alpha and beta from cytosol to membrane as well as the PKC enzymatic activity. Similarly, the subcellular distribution of the receptor for activated C kinase (RACK), an anchoring protein essential to PKC translocation, was also found to be reduced in EBV-infected monocytes. Transfection of 293T cells with an expression vector coding for the immediate-early protein ZEBRA of EBV resulted in impaired PMA-induced translocation and activity of PKC. Using co-immunoprecipitation assays, the ZEBRA protein was found to physically interact with the RACK1 protein. Thus interaction of ZEBRA with RACK likely results in the inhibition of PKC activity, which in turn affects functions of monocytes, such as phagocytosis.
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Affiliation(s)
- Melanie Tardif
- Laboratory of Viral Immunology, Laboratory of Virology, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier de l'Université Laval, and Université Laval, Québec G1V 4G2, Canada
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11
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Yoshioka M, Ishiguro N, Ishiko H, Ma X, Kikuta H, Kobayashi K. Heterogeneous, restricted patterns of Epstein-Barr virus (EBV) latent gene expression in patients with chronic active EBV infection. J Gen Virol 2001; 82:2385-2392. [PMID: 11562532 DOI: 10.1099/0022-1317-82-10-2385] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Epstein-Barr virus (EBV) has been shown to infect T lymphocytes and to be associated with a chronic active infection (CAEBV), which has been recognized as a mainly non-neoplastic T-cell lymphoproliferative disorder (T-cell LPD). The systemic distribution of EBV genomes was studied, by real-time PCR, in multiple tissues from six patients with CAEBV, including three patients with T-cell LPD, one patient with B-cell LPD and two patients with undetermined cell-type LPD. There were extremely high loads of EBV genomes in all tissues from the patients. This reflects an abundance of circulating and infiltrating EBV-infected cells and a wide variety of clinical symptoms in the affected tissues. We chose one sample from each patient that was shown by real-time PCR to contain a high load of EBV genomes and examined the expression of EBV latent genes by RT-PCR. EBER1 and EBNA1 transcripts were detected in all samples. Only one sample also expressed EBNA2, LMP1 and LMP2A transcripts in addition to EBER1 and EBNA1 transcripts. Two of the remaining five samples expressed LMP1 and LMP2A transcripts. One sample expressed LMP2A but not LMP1 and EBNA2 transcripts. Another sample expressed EBNA2 but not LMP1 and LMP2A transcripts. The other sample did not express transcripts of any of the other EBNAs or LMPs. None of the samples expressed the viral immediate-early gene BZLF1. These results showed that EBV latent gene expression in CAEBV is heterogeneous and that restricted forms of EBV latency might play a pathogenic role in the development of CAEBV.
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Affiliation(s)
- Mikio Yoshioka
- Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan1
| | - Nobuhisa Ishiguro
- Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan1
| | - Hiroaki Ishiko
- Department of Infectious Diseases, Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan2
| | - Xiaoming Ma
- Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan1
| | - Hideaki Kikuta
- Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan1
| | - Kunihiko Kobayashi
- Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan1
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12
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Stable expression of Epstein-Barr virus BZLF-1–encoded ZEBRA protein activates p53-dependent transcription in human Jurkat T-lymphoblastoid cells. Blood 2000. [DOI: 10.1182/blood.v96.2.625.014k27_625_634] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Interaction between viral proteins and tumor suppressor p53 is a common mechanism of viral pathogenesis. The Epstein-Barr virus (EBV) BZLF-1 ORF-encoded ZEBRA protein (also denoted EB1, Z, Zta) binds to p53 in vitro and has been associated with the altered transcription of p53-regulated genes in B lymphocytes and epithelial cells. In this work, Jurkat T-lymphoblastoid cells that express ZEBRA were characterized by the use of transiently transfected p53 and p53 reporter genes. Stable expression of ZEBRA was associated with the activation of p53-dependent transcription and increased p53 dependent apoptotic cell death. In Jurkat cell lines, stably expressed ZEBRA protein was apparently localized to the cell cytoplasm, in contrast to the typical nuclear localization of this protein in other cell types. Previous studies have suggested that EBV infection of T lymphocytes may contribute to the malignant transformation of T cells and the increased replication of human immunodeficiency virus. Our observations suggest a mechanism through which ZEBRA protein expressed in human T lymphocytes could alter T-cell proliferation and apoptosis during EBV infection.
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Stable expression of Epstein-Barr virus BZLF-1–encoded ZEBRA protein activates p53-dependent transcription in human Jurkat T-lymphoblastoid cells. Blood 2000. [DOI: 10.1182/blood.v96.2.625] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractInteraction between viral proteins and tumor suppressor p53 is a common mechanism of viral pathogenesis. The Epstein-Barr virus (EBV) BZLF-1 ORF-encoded ZEBRA protein (also denoted EB1, Z, Zta) binds to p53 in vitro and has been associated with the altered transcription of p53-regulated genes in B lymphocytes and epithelial cells. In this work, Jurkat T-lymphoblastoid cells that express ZEBRA were characterized by the use of transiently transfected p53 and p53 reporter genes. Stable expression of ZEBRA was associated with the activation of p53-dependent transcription and increased p53 dependent apoptotic cell death. In Jurkat cell lines, stably expressed ZEBRA protein was apparently localized to the cell cytoplasm, in contrast to the typical nuclear localization of this protein in other cell types. Previous studies have suggested that EBV infection of T lymphocytes may contribute to the malignant transformation of T cells and the increased replication of human immunodeficiency virus. Our observations suggest a mechanism through which ZEBRA protein expressed in human T lymphocytes could alter T-cell proliferation and apoptosis during EBV infection.
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14
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Savard M, Bélanger C, Tardif M, Gourde P, Flamand L, Gosselin J. Infection of primary human monocytes by Epstein-Barr virus. J Virol 2000; 74:2612-9. [PMID: 10684275 PMCID: PMC111749 DOI: 10.1128/jvi.74.6.2612-2619.2000] [Citation(s) in RCA: 95] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolated nuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This was supported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two early lytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with the replicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were also detected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negative results. Viral replication was also confirmed by the release of newly synthesized infectious viral particles in supernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reduced phagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads. Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functions might represent a new mechanism to disrupt the immune response and promote viral propagation during the early stages of infection.
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Affiliation(s)
- M Savard
- Laboratory of Viral Immunology, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Université Laval, Sainte-Foy, Québec, Canada
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Dreyfus DH, Nagasawa M, Pratt JC, Kelleher CA, Gelfand EW. Inactivation of NF-κB by EBV BZLF-1-Encoded ZEBRA Protein in Human T Cells. THE JOURNAL OF IMMUNOLOGY 1999. [DOI: 10.4049/jimmunol.163.11.6261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Abstract
We have previously shown that the EBV ZEBRA protein (also denoted EB1, Z, or Zta) encoded by the BZLF open reading frame is expressed in primary human thymocytes and in human T lymphoblastoid cell lines infected by EBV. Expression of EBV-encoded gene products in T lymphocytes could contribute to viral pathogenesis during acute EBV infection as well as in individuals coinfected with EBV and HIV. HPB-ALL and Jurkat T lymphoblastoid cell lines transiently and stably expressing ZEBRA were characterized in this work. Expression of ZEBRA protein in human T lymphoblastoid cells was associated with decreased expression of an NF-κB reporter gene, altered expression of the NF-κB p50 protein subunit, and decreased DNA binding by components of NF-κB. These observations suggest that inactivation of NF-κB transcription by ZEBRA in EBV-infected T cells may be a novel mechanism of viral pathogenesis analogous in part to over-expression of the endogenous cytoplasmic inhibitor of NF-κB, IκBα.
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Affiliation(s)
- David H. Dreyfus
- Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
| | - Masayuki Nagasawa
- Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
| | - Joanne C. Pratt
- Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
| | - Colm A. Kelleher
- Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
| | - Erwin W. Gelfand
- Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Paterson RK, Bluethmann H, Tseng P, Dunlap A, Finkel TH. Development and function of autospecific dual TCR+ T lymphocytes. Int Immunol 1999; 11:113-9. [PMID: 10050679 DOI: 10.1093/intimm/11.1.113] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Recent studies have challenged the long held concept that each T lymphocyte expresses on its surface only a single, unique alphabetaTCR. Dual TCR+ T cells have been recognized, however, their origin and potential to escape screening for self-reactivity remain obscure. We now report the thymic generation of dual alphabetaTCR+ T cells in the H-2Db/H-Y-specific TCR transgenic (Tg) mouse. Dual TCR+ thymocytes were positively selected less efficiently than single TCR+ thymocytes, although a subset attained maturity. Importantly, when TCR Tg mice were bred onto a negatively selecting background, auto-specific cells survived central deletion and matured as CD4+ dual TCR+ cells. These cells were autoreactive when CD8 expression was restored. The existence of autospecific, dual TCR+ T cells may have implications for the maintenance of self tolerance.
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Affiliation(s)
- R K Paterson
- Department of Immunology, University of Colorado Health Sciences Center, Denver 80262, USA
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Abstract
The role of neutrophils during Epstein-Barr virus (EBV) infection is not known. Disruption of the initial and nonspecific immune response may favor the spread of EBV infection. We have previously shown that EBV interacts with human neutrophils and modulates protein expression. In this study we have investigated the ability of EBV to infect neutrophils. Electron microscopy studies showed penetration of virus and its subsequent localization to the nucleus. The presence of viral genomes in isolated nuclei from neutrophils was also shown by polymerase chain reaction (PCR). Expression of viral transcripts like EBNA-2 (Epstein-Barr nuclear antigen-2) and ZEBRA (BamHI Z EBV replication activator) was not detected by reverse transcriptase (RT)-PCR, suggesting that EBV does not seem to establish a latent or a lytic infection in neutrophils. However, at 20 hours post-EBV infection, 77% of cells were apoptotic as compared to 22% in uninfected cell cultures, as evaluated by flow cytometry. This EBV-induced apoptosis was prevented by the addition of granulocyte-macrophage colony-stimulating factor to the cell cultures. Apoptotic cell death seems to implicate the Fas/Fas ligand (L) pathway, as reflected by an increase of Fas/Fas L expression on neutrophils treated with EBV and an increase of soluble Fas L, which may function in an autocrine/paracrine pathway to mediate cell death. Lastly, EBV genome was detected from neutrophils of infectious mononucleosis (IM) patients in contrast to neutrophils obtained from healthy EBV-seropositive donors. Our findings on the interactions of EBV with neutrophils will then provide new insights on the immunosuppressive effects associated with EBV infection.
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Nakamura H, Iwakiri D, Ono Y, Fujiwara S. Epstein-Barr-virus-infected human T-cell line with a unique pattern of viral-gene expression. Int J Cancer 1998; 76:587-94. [PMID: 9590138 DOI: 10.1002/(sici)1097-0215(19980518)76:4<587::aid-ijc23>3.0.co;2-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Epstein-Barr-virus(EBV)-gene expression was analyzed in clonal sub-lines of the human T-cell line MT-2 that are persistently infected by the virus with a positive selection marker. Immunoblot analyses showed the expression of EBV proteins associated with the latent viral cycle, including the EBV nuclear antigen 1 (EBNA1) and the latent membrane protein 1 (LMP1), whereas EBNA2 was not detected. RT-PCR further demonstrated the expression of LMP2A, EBV-encoded small RNA 1 (EBER1), and complementary-strand transcripts from the BamHI-A fragment. EBNA2 mRNA was detected at a very low level. In addition to these latent viral functions, the BZLF1 protein, a transcription factor that activates a cascade of replicative-gene expression in EBV-infected B cells, was detected in these MT-2 clones by immunoblot analysis and immuno-enzymatic staining. Moreover, mRNA from other genes of the early virus-replicative cycle, such as BRLF1, BSMLF1, and BALF2, as well as BZLF1 was detected by Northern-blot analysis. Since sub-lines of the human B-cell lines BJAB and Louckes infected with similar recombinant EBV did not express these early genes, these data suggest that MT-2 cells are relatively permissive for expression of early EBV-replicative functions.
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Affiliation(s)
- H Nakamura
- Department of Microbiology, Nihon University School of Medicine, Tokyo, Japan
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19
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A Syndrome of Peripheral Blood T-Cell Infection With Epstein-Barr Virus (EBV) Followed by EBV–Positive T-Cell Lymphoma. Blood 1998. [DOI: 10.1182/blood.v91.6.2085] [Citation(s) in RCA: 78] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Abstract
The role of Epstein-Barr virus (EBV) in the pathogenesis of severe, chronic active EBV infection and its complications is unclear. We investigated two Japanese patients diagnosed with severe, chronic active EBV infection who subsequently developed EBV–positive T-cell lymphoma. The patients displayed abnormally high antibody titers to EBV antigens, and had evidence of peripheral blood CD4+T-cell infection with EBV, 19 months and 3 months, respectively, before the diagnosis of EBV–positive T-cell lymphoma. The lymphomas were infected with monoclonal EBV and expressed the EBV latency genes EBNA-1, LMP-1, and LMP-2. Genetic studies showed that the virus detected in the T-cell lymphoma was indistinguishable, with respect to type and previously defined LMP-1 and EBNA-1 gene variations, from virus detected in the peripheral blood T cells 19 months earlier. These studies support an important pathogenetic role of T-cell infection with EBV in chronic active EBV infection and in the EBV–positive T-cell lymphoma that followed.
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20
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A Syndrome of Peripheral Blood T-Cell Infection With Epstein-Barr Virus (EBV) Followed by EBV–Positive T-Cell Lymphoma. Blood 1998. [DOI: 10.1182/blood.v91.6.2085.2085_2085_2091] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The role of Epstein-Barr virus (EBV) in the pathogenesis of severe, chronic active EBV infection and its complications is unclear. We investigated two Japanese patients diagnosed with severe, chronic active EBV infection who subsequently developed EBV–positive T-cell lymphoma. The patients displayed abnormally high antibody titers to EBV antigens, and had evidence of peripheral blood CD4+T-cell infection with EBV, 19 months and 3 months, respectively, before the diagnosis of EBV–positive T-cell lymphoma. The lymphomas were infected with monoclonal EBV and expressed the EBV latency genes EBNA-1, LMP-1, and LMP-2. Genetic studies showed that the virus detected in the T-cell lymphoma was indistinguishable, with respect to type and previously defined LMP-1 and EBNA-1 gene variations, from virus detected in the peripheral blood T cells 19 months earlier. These studies support an important pathogenetic role of T-cell infection with EBV in chronic active EBV infection and in the EBV–positive T-cell lymphoma that followed.
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21
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Altmeyer A, Simmons RC, Krajewski S, Reed JC, Bornkamm GW, Chen-Kiang S. Reversal of EBV immortalization precedes apoptosis in IL-6-induced human B cell terminal differentiation. Immunity 1997; 7:667-77. [PMID: 9390690 DOI: 10.1016/s1074-7613(00)80387-8] [Citation(s) in RCA: 53] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Cell death in B cell terminal differentiation rapidly follows cell cycle arrest in IL-6 differentiation of EBV-immortalized, IgG-bearing human lymphoblastoid cells in vitro. G1 arrest is now found to coincide with repression of EBNA2 and LMP1, two EBV genes essential for B cell transformation, without activation of the viral lytic cycle. IL-6-differentiated B cells die by apoptosis, as evidenced by increases in Annexin V binding activity, PARP cleavage, and chromatin disorganization. Expression of Mcl-1, a Bcl-2 family member, was specifically induced during IL-6 differentiation and down-regulated during apoptosis. Thus, IL-6 reverses EBV immortalization and activates the terminal differentiation program in IgG-bearing human B lymphoblastoid cells, including regulation of an anti-apoptotic gene to coordinate differentiation, cell cycle arrest, and cell death.
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Affiliation(s)
- A Altmeyer
- Department of Pathology, Cornell University Medical College, New York, New York 10021, USA
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22
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Knecht H, Berger C, al-Homsi AS, McQuain C, Brousset P. Epstein-Barr virus oncogenesis. Crit Rev Oncol Hematol 1997; 26:117-35. [PMID: 9298328 DOI: 10.1016/s1040-8428(97)00016-4] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Affiliation(s)
- H Knecht
- LINK Laboratories, University of Massachusetts Medical Center, Division of Hematology/Oncology, Worcester, USA
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23
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Abstract
AbstractEpstein-Barr virus (EBV) is a human lymphotropic virus whose main targets have traditionally been described as B lymphocytes and epithelial cells. Here we report the isolation and characterization of largely monoclonal transformed human T-cell lines infected by EBV. The transformed T cells expressed CD2, CD3, and either CD4 or CD8 surface molecules and more generally displayed the phenotype of naive T cells with a complete and clonal rearrangement of the T-cell receptor. None of the cell lines expressed B cells, natural killer, or myeloid antigens or had immunoglobulins genes rearrangement. They grew in the absence of growth factor; however, they all secreted interleukin-2 after mitogenic activation. Polymerase chain reaction (PCR) analysis showed the presence of EBV DNA in all these cell lines. Moreover, Southern blot analysis of one of these cell lines shows the presence of circular episomic EBV DNA, and by Northern blot or reverse transcriptase-PCR analysis, only the expression of Epstein-Barr nuclear antigen-1 (EBNA-1) and latent membrane protein-1 (LMP-1) genes was detected. Finally, the complete transformed phenotype of this T-cell line was shown by its injection into nude or recombination activating gene 2 (RAG2)-deficient mice that led to the formation of solid tumors.
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Dreyfus DH, Kelleher CA, Jones JF, Gelfand EW. Epstein-Barr virus infection of T cells: implications for altered T-lymphocyte activation, repertoire development and autoimmunity. Immunol Rev 1996; 152:89-110. [PMID: 8930669 DOI: 10.1111/j.1600-065x.1996.tb00912.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Affiliation(s)
- D H Dreyfus
- Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA
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