scholarly journals Protein Kinase C-Independent Activation of the Epstein-Barr Virus Lytic Cycle

2002 ◽  
Vol 76 (11) ◽  
pp. 5612-5626 ◽  
Author(s):  
Lyndle Gradoville ◽  
David Kwa ◽  
Ayman El-Guindy ◽  
George Miller
Keyword(s):  
Phorbol Ester ◽  
Epstein Barr Virus ◽  
Hdac Inhibitors ◽  
Lytic Cycle ◽  
Early Gene ◽  
Immediate Early ◽  
Barr Virus ◽  
Kinase C ◽  
Epstein Barr

ABSTRACT The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by n-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.

scholarly journals Microtubule depolymerization activates the Epstein–Barr virus lytic cycle through protein kinase C pathways in nasopharyngeal carcinoma cells

2013 ◽  
Vol 94 (12) ◽  
pp. 2750-2758 ◽  
Author(s):  
Yi-Ru Liu ◽  
Sheng-Yen Huang ◽  
Jen-Yang Chen ◽  
Lily Hui-Ching Wang
Keyword(s):  
Protein Kinase C ◽  
Life Cycle ◽  
Protein Kinase ◽  
Nasopharyngeal Carcinoma ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Kinase C ◽  
Epstein Barr ◽  
In Cells

Elevated levels of antibodies against Epstein–Barr virus (EBV) and the presence of viral DNA in plasma are reliable biomarkers for the diagnosis of nasopharyngeal carcinoma (NPC) in high-prevalence areas, such as South-East Asia. The presence of these viral markers in the circulation suggests that a minimal level of virus reactivation may have occurred in an infected individual, although the underlying mechanism of reactivation remains to be elucidated. Here, we showed that treatment with nocodazole, which provokes the depolymerization of microtubules, induces the expression of two EBV lytic cycle proteins, Zta and EA-D, in EBV-positive NPC cells. This effect was independent of mitotic arrest, as viral reactivation was not abolished in cells synchronized at interphase. Notably, the induction of Zta by nocodazole was mediated by transcriptional upregulation via protein kinase C (PKC). Pre-treatment with inhibitors for PKC or its downstream signalling partners p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) abolished the nocodazole-mediated induction of Zta and EA-D. Interestingly, the effect of nocodazole, as well as colchicine and vinblastine, on lytic gene expression occurred only in NPC epithelial cells but not in cells derived from lymphocytes. These results establish a novel role of microtubule integrity in controlling the EBV life cycle through PKC and its downstream pathways, which represents a tissue-specific mechanism for controlling the life-cycle switch of EBV.

scholarly journals The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

2000 ◽  
Vol 74 (7) ◽  
pp. 3235-3244 ◽  
Author(s):  
Antonella Farina ◽  
Roberta Santarelli ◽  
Roberta Gonnella ◽  
Roberto Bei ◽  
Raffaella Muraro ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Particle Identification ◽  
Early Gene ◽  
Cell Fractionation ◽  
Barr Virus ◽  
F Region ◽  
Epstein Barr

ABSTRACT Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

scholarly journals Identification of Protein Tyrosine Kinases Required for B-Cell- Receptor-Mediated Activation of an Epstein-Barr Virus Immediate-Early Gene Promoter

2004 ◽  
Vol 78 (16) ◽  
pp. 8543-8551 ◽  
Author(s):  
Sandra Lavens ◽  
Emmanuel A. Faust ◽  
Fang Lu ◽  
Michele Jacob ◽  
Messele Leta ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cell ◽  
Tyrosine Kinases ◽  
Epstein Barr Virus ◽  
Cell Receptor ◽  
Lytic Cycle ◽  
Protein Tyrosine Kinases ◽  
Early Gene ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.

scholarly journals Induction of Epstein-Barr Virus Latent Membrane Protein 1 by a Lytic Transactivator Rta

2004 ◽  
Vol 78 (23) ◽  
pp. 13028-13036 ◽  
Author(s):  
Yao Chang ◽  
Heng-Huan Lee ◽  
Shih-Shin Chang ◽  
Tsuey-Ying Hsu ◽  
Pei-Wen Wang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Latent Membrane Protein 1 ◽  
Virus Reactivation ◽  
Barr Virus ◽  
Lmp1 Expression ◽  
Epstein Barr

ABSTRACT Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a transforming protein that affects multiple cell signaling pathways and contributes to EBV-associated oncogenesis. LMP1 can be expressed in some states of EBV latency, and significant induction of full-length LMP1 is also observed frequently during virus reactivation into the lytic cycle. It is still unknown how LMP1 expression is regulated during the lytic stage and whether any EBV lytic protein is involved in the induction of LMP1. In this study, we first identified that LMP1 expression is associated with the spontaneous virus reactivation in EBV-infected 293 cells and that its expression is a downstream event of the lytic cycle. We further found that LMP1 can be induced by ectopic expression of Rta, an EBV immediate-early lytic protein. The Rta-mediated LMP1 induction is independent of another immediate-early protein, Zta. Northern blotting and reverse transcription-PCR analysis revealed that Rta upregulates LMP1 at the RNA level. Reporter gene assays further demonstrated that Rta activates both the proximal and distal promoters of the LMP1 gene in EBV-negative cells. Both the amino and carboxyl termini of the Rta protein are required for the induction of LMP1. In addition, Rta transactivates LMP1 not only in epithelial cells but also in B-lymphoid cells. This study reveals a new mechanism to upregulate LMP1 expression, expanding the knowledge of LMP1 regulation in the EBV life cycle. Considering an equivalent case of Kaposi's sarcoma-associated herpesvirus, induction of a transforming membrane protein by a key lytic transactivator during virus reactivation is likely to be a conserved event for gammaherpesviruses.

scholarly journals Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by InhibitingBAD-Mediatedcaspase-3-Dependent Apoptosis

2015 ◽  
Vol 90 (3) ◽  
pp. 1359-1368 ◽  
Author(s):  
Hyoji Kim ◽  
Hoyun Choi ◽  
Suk Kyeong Lee
Keyword(s):  
Epstein Barr Virus ◽  
Caspase 3 ◽  
Immune Surveillance ◽  
Virus Production ◽  
Lytic Cycle ◽  
Host Cells ◽  
Immediate Early ◽  
Progeny Virus ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and thein vivotriggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genesBRLF1andBZLF1as well asBcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels ofBRLF1andBZLF1were suppressed in cells followingBADknockdown and increased afterBADoverexpression. Progeny virus production was also downregulated by specific knockdown ofBAD. Our results demonstrated thatcaspase-3-dependent apoptosis is a prerequisite forBAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibitingBAD-mediatedcaspase-3-dependent apoptosis, which would trigger immediate early gene expression.IMPORTANCEEBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressingBAD-inducedcaspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

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