scholarly journals Epstein-Barr Virus Protein Kinase BGLF4 Is a Virion Tegument Protein That Dissociates from Virions in a Phosphorylation-Dependent Process and Phosphorylates the Viral Immediate-Early Protein BZLF1

2006 ◽  
Vol 80 (11) ◽  
pp. 5125-5134 ◽  
Author(s):  
Risa Asai ◽  
Ai Kato ◽  
Kentaro Kato ◽  
Mikiko Kanamori-Koyama ◽  
Ken Sugimoto ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Tegument Protein ◽  
Virus Protein ◽  
Barr Virus ◽  
Early Protein ◽  
Dependent Process ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl2 to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

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

2007 ◽  
Vol 81 (21) ◽  
pp. 11850-11860 ◽  
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.

scholarly journals Disruption of Epstein-Barr Virus Latency in the Absence of Phosphorylation of ZEBRA by Protein Kinase C

2002 ◽  
Vol 76 (22) ◽  
pp. 11199-11208 ◽  
Author(s):  
Ayman S. El-Guindy ◽  
Lee Heston ◽  
Yoshimi Endo ◽  
Myung-Sam Cho ◽  
George Miller
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Barr Virus ◽  
Kinase C ◽  
Epstein Barr

ABSTRACT ZEBRA protein converts Epstein-Barr virus (EBV) infection from the latent to the lytic state. The ability of ZEBRA to activate this switch is strictly dependent on the presence of serine or threonine at residue 186 of the protein (A. Francis, T. Ragoczy, L. Gradoville, A. El-Guindy, and G. Miller, J. Virol. 72:4543-4551, 1999). We investigated whether phosphorylation of ZEBRA protein at this site by a serine-threonine protein kinase was required for activation of an early lytic cycle viral gene, BMRF1, as a marker of disruption of latency. Previous studies suggested that phosphorylation of ZEBRA at S186 by protein kinase C (PKC) activated the protein (M. Baumann, H. Mischak, S. Dammeier, W. Kolch, O. Gires, D. Pich, R. Zeidler, H. J. Delecluse, and W. Hammerschmidt, J. Virol 72:8105-8114, 1998). Two residues of ZEBRA, T159 and S186, which fit the consensus for phosphorylation by PKC, were phosphorylated in vitro by this enzyme. Several isoforms of PKC (α, β1, β2, γ, δ, and ε) phosphorylated ZEBRA. All isoforms that phosphorylated ZEBRA in vitro were blocked by bisindolylmaleimide I, a specific inhibitor of PKC. Studies in cell culture showed that phosphorylation of T159 was not required for disruption of latency in vivo, since the T159A mutant was fully functional. Moreover, the PKC inhibitor did not block the ability of ZEBRA expressed from a transfected plasmid to activate the BMRF1 downstream gene. Of greatest importance, in vivo labeling with [32P]orthophosphate showed that the tryptic phosphopeptide maps of wild-type ZEBRA, Z(S186A), and the double mutant Z(T159A/S186A) were identical. Although ZEBRA is a potential target for PKC, in the absence of PKC agonists, ZEBRA is not constitutively phosphorylated in vivo by PKC at T159 or S186. Phosphorylation of ZEBRA by PKC is not essential for the protein to disrupt EBV latency.

scholarly journals Epstein–Barr virus protein kinase BGLF4 interacts with viral transactivator BZLF1 and regulates its transactivation activity

2009 ◽  
Vol 90 (7) ◽  
pp. 1575-1581 ◽  
Author(s):  
Risa Asai ◽  
Ai Kato ◽  
Yasushi Kawaguchi
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Stable Complex ◽  
Virus Protein ◽  
Serine Residue ◽  
Barr Virus ◽  
Viral Transactivator ◽  
Epstein Barr ◽  
Physiological Substrates

BGLF4 is a serine/threonine protein kinase encoded by Epstein–Barr virus. One of the physiological substrates of BGLF4 is viral transactivator BZLF1. In the present study, it was demonstrated that alanine substitution of the serine residue at position 209 (S209A) in BZLF1 eliminated phosphorylation of the protein by BGLF4 in vitro. The S209A mutation in BZLF1, as well as a K102I mutation in BGLF4, which inactivated catalytic activity of the viral kinase, also inhibited formation of a stable BGLF4–BZLF1 complex and downregulation of BZLF1 autotransactivation activity mediated by BGLF4. These results indicate that formation of a stable complex of BGLF4–BZLF1 enables downregulation of BZLF1 autoregulation activity and it appears that BGLF4 phosphorylation of BZLF1 may be involved in these processes.

scholarly journals Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Promotes EBV Reactivation through Activation of the p38 Mitogen-Activated Protein Kinase

2015 ◽  
Vol 90 (2) ◽  
pp. 1129-1138 ◽  
Author(s):  
XueQiao Liu ◽  
Jeffrey I. Cohen
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Epstein Barr Virus ◽  
Virus Production ◽  
Mitogen Activated Protein Kinase ◽  
Tegument Protein ◽  
Barr Virus ◽  
Ebv Reactivation ◽  
Mitogen Activated Protein ◽  
Epstein Barr

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.

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

2009 ◽  
Vol 83 (21) ◽  
pp. 10877-10891 ◽  
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.

Transient Immunodeficiency During Asymptomatic Epstein-Barr Virus Infection

PEDIATRICS ◽  
1983 ◽  
Vol 71 (6) ◽  
pp. 964-967
Author(s):  
THOMAS J. BOWEN ◽  
RALPH J. WEDGWOOD ◽  
HANS D. OCHS ◽  
WERNER HENLE
Keyword(s):  
Epstein Barr Virus ◽  
Mononuclear Cells ◽  
Epstein Barr Virus Infection ◽  
Peripheral Blood Mononuclear ◽  
Immunoglobulin Synthesis ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

In vivo and in vitro humoral and cellular immune responses were studied in a 2½-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus. During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells. In vitro mitogen transformation of lymphocytes was reduced. In vivo antibody responses to the T cell-dependent antigens bacteriophage φX 174 and Keyhole limpet hemocyanin were markedly depressed. These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis.

scholarly journals Development of suppressor T lymphocytes for Epstein-Barr virus-induced B-lymphocyte outgrowth during acute infectious mononucleosis: assessment by two quantitative systems

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 510-517 ◽  
Author(s):  
RT Schooley ◽  
BF Haynes ◽  
J Grouse ◽  
C Payling-Wright ◽  
AS Fauci ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
B Lymphocyte ◽  
Acute Stage ◽  
Cell Mediated Immunity ◽  
Barr Virus ◽  
Epstein Barr

Abstract A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

scholarly journals Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

2006 ◽  
Vol 81 (3) ◽  
pp. 1195-1208 ◽  
Author(s):  
Chih-Chung Lu ◽  
Ho-Ting Huang ◽  
Jiin-Tarng Wang ◽  
Geir Slupphaug ◽  
Tsai-Kun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Efficient Production ◽  
Transcription Activator ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr ◽  
Lytic Dna Replication

ABSTRACT Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

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