Protein kinase inhibitors that inhibit induction of lytic program and replication of Epstein–Barr virus

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-κB promoter activity in AP-1– and NF-κB–luciferase reporter assays. We found that EBV BGLF2 activated AP-1 but not NF-κB reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK), both of which are important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in latently EBV-infected lymphoblastoid cell lines, and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinases 1 and 2 (MEK1/2) reduced expression of BZLF1 and virus production induced by BGLF2. In summary, the EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production.IMPORTANCEEpstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression or inhibition of p38/MAPK, which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein which is delivered to cells upon infection activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

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The Epstein-Barr Virus Protein Kinase BGLF4 and the Exonuclease BGLF5 Have Opposite Effects on the Regulation of Viral Protein Production

Journal of Virology ◽

10.1128/jvi.00525-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 10877-10891 ◽

Cited By ~ 29

Author(s):

Regina Feederle ◽

Anja M. Mehl-Lautscham ◽

Helmut Bannert ◽

Henri-Jacques Delecluse

Keyword(s):

Protein Kinase ◽

Viral Protein ◽

Epstein Barr Virus ◽

Protein Production ◽

Opposite Effect ◽

Viral Gene ◽

Virus Protein ◽

Barr Virus ◽

Viral Genes ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus BGLF4 and BGLF5 genes encode a protein kinase and an alkaline exonuclease, respectively. Both proteins were previously found to regulate multiple steps of virus replication, including lytic DNA replication and primary egress. However, while inactivation of BGLF4 led to the downregulation of several viral proteins, the absence of BGLF5 had the opposite effect. Using recombinant viruses that lack both viral enzymes, we confirm and extend these initial observations, e.g., by showing that both BGLF4 and BGLF5 are required for proper phosphorylation of the DNA polymerase processivity factor BMRF1. We further found that neither BGLF4 nor BGLF5 is required for baseline viral protein production. Complementation with BGLF5 downregulated mRNA levels and translation of numerous viral genes, though to various degrees, whereas BGLF4 had the opposite effect. BGLF4 and BGLF5 influences on viral expression were most pronounced for BFRF1 and BFLF2, two proteins essential for nuclear egress. For most viral genes studied, cotransfection of BGLF4 and BGLF5 had only a marginal influence on their expression patterns, showing that BGLF4 antagonizes BGLF5-mediated viral gene shutoff. To be able to exert its functions on viral gene expression, BGLF4 must be able to escape BGLF5's shutoff activities. Indeed, we found that BGLF5 stimulated the BGLF4 gene's transcription through an as yet uncharacterized molecular mechanism. The BGLF4/BGLF5 enzyme pair builds a regulatory loop that allows fine-tuning of virus protein production, which is required for efficient viral replication.

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A role for protein kinase PKR in the mediation of Epstein–Barr virus latent membrane protein-1-induced IL-6 and IL-10 expression

Cytokine ◽

10.1016/j.cyto.2010.01.008 ◽

2010 ◽

Vol 50 (2) ◽

pp. 210-219 ◽

Cited By ~ 5

Author(s):

San San Lin ◽

Davy C.W. Lee ◽

Anna H.Y. Law ◽

Jun Wei Fang ◽

Daniel T.T. Chua ◽

...

Keyword(s):

Protein Kinase ◽

Membrane Protein ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Latent Membrane Protein 1 ◽

Barr Virus ◽

Epstein Barr ◽

Virus Latent Membrane Protein

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Regulation of the Double-stranded RNA-Dependent Protein Kinase PKR by RNAs Encoded by a Repeated Sequence in the Epstein-Barr Virus Genome

Nucleic Acids Research ◽

10.1093/nar/24.22.4471 ◽

1996 ◽

Vol 24 (22) ◽

pp. 4471-4478 ◽

Cited By ~ 31

Author(s):

A. Elia ◽

K. G. Laing ◽

A. Schofield ◽

V. J. Tilleray ◽

M. J. Clemens

Keyword(s):

Protein Kinase ◽

Epstein Barr Virus ◽

Repeated Sequence ◽

Virus Genome ◽

Dependent Protein Kinase ◽

Double Stranded Rna ◽

Barr Virus ◽

Dependent Protein ◽

Epstein Barr

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

Journal of General Virology ◽

10.1099/vir.0.058040-0 ◽

2013 ◽

Vol 94 (12) ◽

pp. 2750-2758 ◽

Cited By ~ 8

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.

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Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase

Journal of Virology ◽

10.1128/jvi.76.18.9556-9561.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9556-9561 ◽

Cited By ~ 60

Author(s):

Shao-Yin Chen ◽

Jean Lu ◽

Yin-Chu Shih ◽

Ching-Hwa Tsai

Keyword(s):

Protein Kinase ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Erk Pathway ◽

Latent Membrane Protein 2A ◽

Extracellular Signal Regulated Kinase ◽

Barr Virus ◽

Extracellular Signal ◽

Infected Cells ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in both EBV-infected cells and EBV-associated malignancies. However, the function of LMP2A is still veiled. In this study, LMP2A was found to induce the kinase activities of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK. Furthermore, the downstream effector c-Jun showed hyperphosphorylation under LMP2A expression. The phosphorylation could be inhibited by the ERK pathway inhibitor PD98059, indicating that ERK may contribute to the phosphorylation of c-Jun in LMP2A-expressing cells. The impact on c-Jun phosphorylation by mitogen-activated protein kinase (MAPK) is suggested to increase c-Jun protein stability, and this was also observed in LMP2A-expressing cells by a protein synthesis inhibition assay. Moreover, LMP2A-induced cell invasion was inhibited in the presence of the ERK pathway inhibitor. Taken together, we suggest that LMP2A may exploit MAPK kinases and affect both the phosphorylation and stability of c-Jun protein. Additionally, LMP2A may thereby promote the mobility of the cells. In doing so, it may enhance the mobility of EBV-infected cells and contribute to the metastatic process of malignant cells. Here we demonstrated the first evidence of LMP2A-induced migration and the underlying pathways accounting for it.

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The Epstein-Barr virus bZIP transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin-dependent kinase inhibitors.

The EMBO Journal ◽

10.1002/j.1460-2075.1996.tb00635.x ◽

1996 ◽

Vol 15 (11) ◽

pp. 2748-2759 ◽

Cited By ~ 145

Author(s):

C. Cayrol ◽

E. K. Flemington

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Epstein Barr Virus ◽

Kinase Inhibitors ◽

Bzip Transcription Factor ◽

Cyclin Dependent Kinase ◽

Barr Virus ◽

Cycle Arrest ◽

G1 Cell Cycle Arrest ◽

Epstein Barr

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Protein Kinase CK2 Phosphorylation of EB2 Regulates Its Function in the Production of Epstein-Barr Virus Infectious Viral Particles

Journal of Virology ◽

10.1128/jvi.01421-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11850-11860 ◽

Cited By ~ 21

Author(s):

Cahora Medina-Palazon ◽

Henri Gruffat ◽

Fabrice Mure ◽

Odile Filhol ◽

Valérie Vingtdeux-Didier ◽

...

Keyword(s):

Protein Kinase ◽

Nuclear Export ◽

Epstein Barr Virus ◽

Nuclear Export Signal ◽

Regulatory Subunit ◽

Viral Mrnas ◽

Barr Virus ◽

Early Protein ◽

Epstein Barr

ABSTRACT The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2α and -β subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2β regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293BMLF1-KO cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293BMLF1-KO cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.

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