The major immunogenic epitopes of Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines.

Chen, Mei‐Ru; Tsai, Ching-Hwa; Wu, Fuli; Kan, Shih-hsin; Yang, Czau‐Siung; Chen, Jen‐Yang

doi:10.1099/0022-1317-80-2-447

Cited by 26 publications

(24 citation statements)

References 32 publications

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“…Because most of the polymorphic regions were found to match putative MHC class I binding epitopes, it was suggested that these variations could contribute to the immune escape of EBV in EBV-associated malignant diseases. This hypothesis was supported partly by our finding that the regions of sequence variation of EBNA-1 were dominant immunogenic epitopes in the mouse [Chen et al, 1999].…”

Section: Introductionmentioning

confidence: 56%

EBNA‐1 sequence variations reflect active EBV replication and disease status or quiescentlatency in lymphocytes

Wang

Sheeng

et al. 2003

Journal of Medical Virology

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EBV infects most of the global population, but only a small percentage of infected individuals develop EBV-associated malignancies. Host and viral factors may play a role in determining the clinical outcome. Because EBNA-1 functions as an oriP binding protein and links the episomal viral DNA to metaphase chromosomes, variation of EBNA-1 has been suggested to contribute to determining the tissue tropism of EBV and the development of various EBV-associated diseases. Five subtypes have been described, according to the amino acid at residue 487: P-ala (B95-8 prototype), P-thr (aa 487 ala to thr), V-val, V-pro and V-leu. Other studies, however, concluded that EBNA-1 sequence variation simply reflects the geographical distribution of EBV. To clarify these possibilities, we collected DNA samples from healthy individuals and patients with various EBV-associated diseases in Taiwan for PCR amplification and DNA sequencing. The results indicate that: 1) V-val EBNA-1 was detected in patients with nasopharyngeal carcinoma (NPC) and other EBV-associated malignant diseases; 2) the prototype P-ala strain was detected only in peripheral blood lymphocytes; 3) mixed populations of different subtypes of N-terminal and C-terminal sequences were observed in samples from one patient with nasopharyngeal carcinoma, one with T lymphoma and one with infectious mononucleosis sample; 4) intermediate variations between P-ala and V-val were observed in T-lymphoma, Hodgkin disease and infectious mononucleosis samples; and 5) in comparison with the major sequences identified in healthy carriers, the EBNA-1 sequences in peripheral lymphocytes from nasopharyngeal carcinoma were mixed types in 4 of 5 patients, implying increasing frequency of V-val might correlate with the progression of nasopharyngeal carcinoma.

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Section: Introductionmentioning

confidence: 56%

EBNA‐1 sequence variations reflect active EBV replication and disease status or quiescentlatency in lymphocytes

Wang

Sheeng

et al. 2003

Journal of Medical Virology

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“…An EBNA-1 monoclonal antibody tag system was therefore used to enhance the specificity of the immunoprecipitation reaction. 5C11 recognizes an epitope between amino acids 408 and 446 of EBNA-1, a region which contains no serine or threonine residues, and was shown to react with this sequence in both immunoprecipitation and Western immunoblot assays (8). Therefore, EBNA-1(408-446) and BGLF4 were expressed as a fusion protein using a derivative of pGH254 which contains a T7 RNA promoter and a black beetle virus leader sequence to enhance translation (18).…”

Section: Resultsmentioning

confidence: 99%

“…This is a derivative of pBD7 (18) and contains a T7 promoter sequence and a black beetle virus leader sequence to enhance protein expression levels. The BamHI DNA fragment containing BGLF4 was subcloned into the BglII site of pGH254, and then a PCR product, encoding amino acids 408 to 446 of EBNA-1, was cloned upstream of BGLF4 as a tag for detection and immunoprecipitation with monoclonal antibody 5C11 (8). This construct was designated pSJC12(E1/BGLF4).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Purified BGLF4 was used to immunize New Zealand White rabbits to generate anti-BGLF4 specific antisera. The monoclonal antibody 5C11 specifically recognizes an epitope between amino acids 408 and 446 of EBNA-1 (8). Monoclonal antibodies antiphosphoserine (PSR-45, P3430) and antiphosphothreonine (pTR-8, P-3555) were from Sigma, and antiphosphotyrosine (4G10) was from Upstate Biotechnology.…”

Section: Plasmid Constructionmentioning

confidence: 99%

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A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro

Chen

Chang

Huang

et al. 2000

J Virol

105

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The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.Protein kinases are known to be involved in the regulation of a wide variety of eukaryotic cellular functions including cell metabolism, cell cycle control, hormone response, and control of transcription and translation. Studying viral protein kinases might therefore lead to an understanding of the mechanisms of virus replication and virus-cell interactions. Most of the protein kinases of the retroviruses are Tyr protein kinases, such as v-src and v-erb, which may contribute to the growth transformation phenotype of the virally infected host cells (for a review, see reference 32). The first protein kinase gene demonstrated in a eukaryotic DNA virus was that contained in the unique short (US) regions of the related human and porcine alphaherpesviruses, herpes simplex virus type 1 (HSV-1), and pseudorabies virus (20). Other protein kinases have been reported in DNA viruses, including protein kinase B1 of the poxviruses (45, 46) and ORF9 of baculovirus (42).Phosphorylation of cellular and viral proteins, which has been observed during lytic infection of cells by herpesviruses, seems to be a common phenomenon which involves a number of different protein kinase activities (21). Two groups of viral protein kinase activities, US3 and UL13, have been identified in alphaherpesviruses. The US3 gene of HSV-1 (37) and the VZV66 gene of varicella-zoster virus (VZV) (19) were predicted to encode protein kinases on the basis of their strong similarity to the family of eukaryotic serine/threon...

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“…NP_001562), IRF3Ј lacks part of the IAD (aa 327 to 375) and the transactivation domain (aa 327 to 394) of the IRF3 protein. To confirm the interaction between BGLF4 and IRF3Ј, 293T cells were cotransfected with E1/BGLF4(1-293) and HA-IRF3Ј(282-452) expression constructs and subjected to coimmunoprecipitation with E1 tag-specific MAb 5C11 (13). HA-IRF3Ј(282-452) was coimmunoprecipitated from cell extracts specifically with E1/BGLF4(1-293) (Fig.…”

Section: Interaction Of Bglf4 Kinase With a Cellular Irf3 Variantmentioning

confidence: 98%

Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway

Wang

Doong

Teng

et al. 2009

J Virol

124

115

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The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pulldown in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I: C)-stimulated IFN-␤ promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-␤ mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-␤ promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three prolinedependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.The innate immune response is the first-line defense against viral infection. The production of interferons (IFNs) and other cytokines to prevent virus replication and spread is at the center of the antiviral response and requires the activation of multiple transcription activators. The family of IFN regulatory factors (IRFs) is defined by a highly conserved amino-terminal DNA binding domain (DBD) containing five tryptophan repeats and a unique C-terminal domain, the IRF association domain (IAD) (29). IRF3 and IRF7 are two major direct transducers of virus-mediated signaling that induce type I IFNs. IRF3 is a constitutively expressed phosphoprotein of 427 amino acids, which can shuttle into and out of the nucleus in its inactive form. Upon virus infection, cellular TBK-1-and IKKε-mediated phosphorylation of serines 385 and 386 and the serine/threonine cluster between amino acids 396 and 405 of IRF3 lead to its conformational change and activation (19,29,65).The activated IRF3 then undergoes homodimerization or heterodimerization with IRF7, nuclear localization, and association with the coactivator CBP/P300 (29). The phosphor...

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The major immunogenic epitopes of Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines.

Abstract: Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a protein expressed consistently in EBV-

Cited by 26 publications

References 32 publications

EBNA‐1 sequence variations reflect active EBV replication and disease status or quiescentlatency in lymphocytes

EBNA‐1 sequence variations reflect active EBV replication and disease status or quiescentlatency in lymphocytes

A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro

Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway

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