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PMID: 10708424 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro.

Journal of virology ·Vol. 74 ·No. 7 ·2000-04-00 ·Pages 3093-104

Chen MR, Chang SJ, Huang H, Chen JY

Abstract

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.

MeSH Terms
Amino Acid Sequence Antigens, Viral/metabolism Cloning, Molecular Cytoplasm/enzymology Enzyme Inhibitors/pharmacology Escherichia coli/genetics Heparin/pharmacology Herpesvirus 4, Human/enzymology,genetics Immune Sera Kinetics Molecular Sequence Data Okadaic Acid/pharmacology Phosphorylation Protein Kinases/metabolism Protein Serine-Threonine Kinases/chemistry,genetics,metabolism Sequence Homology, Amino Acid Serine/metabolism Threonine/metabolism Viral Proteins
Chemicals
Antigens, Viral Enzyme Inhibitors Epstein-Barr virus early antigen diffuse component Immune Sera Viral Proteins Okadaic Acid Threonine Serine Heparin Protein Kinases BGLF4 protein, Epstein-Barr virus Protein Serine-Threonine Kinases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Chen M R
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan. [email protected]
Chang S J
Huang H
Chen J Y
References (55)
55 references, click to expand
  1. Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells.
    Virology. 1991 Sep;184(1):330-40 PMID: 1651595
  2. Phosphorylation of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA.
    Virology. 1992 Jun;188(2):916-20 PMID: 1316687
  3. The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase.
    J Biol Chem. 1998 Jan 16;273(3):1435-43 PMID: 9430680
  4. Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).
    Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14862-7 PMID: 8962146
  5. Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses.
    Nucleic Acids Res. 1986 Feb 25;14(4):1765-77 PMID: 3005981
  6. pp60c-src expression is induced by activation of normal human T lymphocytes.
    J Immunol. 1995 Apr 15;154(8):3678-85 PMID: 7535811
  7. Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase.
    J Gen Virol. 1989 May;70 ( Pt 5):1151-60 PMID: 2543772
  8. A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted.
    J Gen Virol. 1993 Mar;74 ( Pt 3):387-95 PMID: 8383174
  9. Protein kinases and phosphatases: regulation by autoinhibitory domains.
    Biotechnol Appl Biochem. 1993 Oct;18 ( Pt 2):185-200 PMID: 8251116
  10. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor.
    Nature. 1987 Aug 27-Sep 2;328(6133):820-3 PMID: 3498122
  11. Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein.
    J Gen Virol. 1994 Feb;75 ( Pt 2):283-93 PMID: 8113749
  12. Viral protein kinases and protein phosphatases.
    Pharmacol Ther. 1993 Sep;59(3):343-89 PMID: 8309996
  13. The PK domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for immediate-early gene expression and virus growth.
    J Virol. 1998 Nov;72(11):9131-41 PMID: 9765459
  14. Human herpesvirus 8-encoded thymidine kinase and phosphotransferase homologues confer sensitivity to ganciclovir.
    J Virol. 1999 Jun;73(6):4786-93 PMID: 10233939
  15. Identification of a target protein of US3 protein kinase of herpes simplex virus type 2.
    J Gen Virol. 1994 Aug;75 ( Pt 8):2065-8 PMID: 8046410
  16. Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.
    Virology. 1992 Sep;190(1):184-92 PMID: 1326802
  17. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification.
    FASEB J. 1995 May;9(8):576-96 PMID: 7768349
  18. Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.
    J Virol. 1994 Mar;68(3):1350-9 PMID: 8107200
  19. Use of bacterially expressed GST/EBNA-1 fusion proteins for detection of antibodies in sera from patients with nasopharyngeal carcinoma and healthy donors.
    Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi. 1996 May;29(2):65-79 PMID: 10592788
  20. pBD7, a novel cell-free expression vector with efficient translation initiation signal.
    Nucleic Acids Res. 1987 May 11;15(9):3933 PMID: 3675735
  21. Serum responses to the combination of Epstein-Barr virus antigens from both latent and acute phases in nasopharyngeal carcinoma: complementary test of EBNA-1 with EA-D.
    Cancer Epidemiol Biomarkers Prev. 1997 May;6(5):363-8 PMID: 9149897
  22. Processing of the herpes simplex virus regulatory protein alpha 22 mediated by the UL13 protein kinase determines the accumulation of a subset of alpha and gamma mRNAs and proteins in infected cells.
    Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6701-5 PMID: 8393574
  23. The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation of the regulatory protein alpha 22.
    Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7310-4 PMID: 1323829
  24. Serine-173 of the Epstein-Barr virus ZEBRA protein is required for DNA binding and is a target for casein kinase II phosphorylation.
    Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10115-9 PMID: 8234266
  25. The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).
    J Biol Chem. 1992 May 15;267(14):9645-53 PMID: 1315764
  26. The US3 protein kinase of herpes simplex virus type 2 is associated with phosphorylation of the UL12 alkaline nuclease in vitro.
    Arch Virol. 1995;140(9):1637-44 PMID: 7487495
  27. Localization of the antigenic sites and intrinsic protein kinase domain within a 300 amino acid segment of the ribonucleotide reductase large subunit from herpes simplex virus type 2.
    Virology. 1992 Mar;187(1):360-7 PMID: 1371028
  28. Enzyme activity dot blots for assaying protein kinases.
    Methods Enzymol. 1991;200:85-90 PMID: 1956342
  29. Human herpesvirus 6 is closely related to human cytomegalovirus.
    J Virol. 1990 Jan;64(1):287-99 PMID: 2152817
  30. Characterization of Monoclonal Antibodies to the Zta and DNase Proteins of Epstein-Barr Virus.
    J Biomed Sci. 1997 Mar-Jun;4(2-3):69-77 PMID: 11725136
  31. Serine protein kinase associated with varicella-zoster virus ORF 47.
    Virology. 1992 Nov;191(1):9-18 PMID: 1329339
  32. Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus.
    J Virol. 1989 Jan;63(1):450-5 PMID: 2535748
  33. The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication.
    J Virol. 1996 Oct;70(10):7312-7 PMID: 8794389
  34. High-efficiency transformation of mammalian cells by plasmid DNA.
    Mol Cell Biol. 1987 Aug;7(8):2745-52 PMID: 3670292
  35. Molecular characterization of a cDNA clone encoding the Epstein-Barr virus (EBV) DNase.
    J Virol Methods. 1990 Aug;29(2):127-41 PMID: 2176660
  36. The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse.
    Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11969-74 PMID: 9751774
  37. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.
    Nature. 1992 Jul 9;358(6382):162-4 PMID: 1319560
  38. 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.
    J Gen Virol. 1999 Feb;80 ( Pt 2):447-55 PMID: 10073707
  39. Polypeptide synthesis and phosphorylation in Epstein-Barr virus-infected cells.
    J Virol. 1980 May;34(2):455-63 PMID: 6246275
  40. The complete DNA sequence of varicella-zoster virus.
    J Gen Virol. 1986 Sep;67 ( Pt 9):1759-816 PMID: 3018124
  41. Characterization of the putative protein kinases specified by varicella-zoster virus genes 47 and 66.
    J Gen Virol. 1994 Feb;75 ( Pt 2):317-26 PMID: 8113753
  42. Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir.
    Nature. 1992 Jul 9;358(6382):160-2 PMID: 1319559
  43. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.
    J Virol. 1992 Jul;66(7):4413-26 PMID: 1602551
  44. Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.
    J Virol. 1987 Sep;61(9):2947-9 PMID: 3039183
  45. CLUSTAL V: improved software for multiple sequence alignment.
    Comput Appl Biosci. 1992 Apr;8(2):189-91 PMID: 1591615
  46. The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein kinase.
    J Gen Virol. 1992 Feb;73 ( Pt 2):303-11 PMID: 1311359
  47. Eukaryotic elongation factor 1delta is hyperphosphorylated by the protein kinase encoded by the U(L)13 gene of herpes simplex virus 1.
    J Virol. 1998 Mar;72(3):1731-6 PMID: 9499021
  48. ICP22 and the UL13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of RNA polymerase II.
    J Virol. 1999 Jul;73(7):5593-604 PMID: 10364308
  49. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
    Nucleic Acids Res. 1994 Nov 11;22(22):4673-80 PMID: 7984417
  50. Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region.
    Virology. 1991 Nov;185(1):229-41 PMID: 1926775
  51. Cellular elongation factor 1delta is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses.
    J Virol. 1999 May;73(5):4456-60 PMID: 10196346
  52. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.
    J Virol. 1990 Feb;64(2):574-83 PMID: 2296077
  53. The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.
    Virology. 1997 Mar 31;230(1):22-34 PMID: 9126259
  54. Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells.
    J Virol. 1992 Sep;66(9):5200-9 PMID: 1323689
  55. Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).
    J Virol. 1989 Aug;63(8):3389-98 PMID: 2545912
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/jvi.74.7.3093-3104.2000
Email
[email protected]
Published
2000-04-00
Pages
3093-104
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC111808
Subset
IM
External Links
PubMed source DOI: 10.1128/jvi.74.7.3093-3104.2000 PMC Full Text View journal details
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