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. 1996 Feb;70(2):1050–1060. doi: 10.1128/jvi.70.2.1050-1060.1996

Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication.

K Xia 1, D M Knipe 1, N A DeLuca 1
PMCID: PMC189911  PMID: 8551563

Abstract

Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.

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Selected References

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  1. Ackermann M., Braun D. K., Pereira L., Roizman B. Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies. J Virol. 1984 Oct;52(1):108–118. doi: 10.1128/jvi.52.1.108-118.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blaho J. A., Roizman B. ICP4, the major regulatory protein of herpes simplex virus, shares features common to GTP-binding proteins and is adenylated and guanylated. J Virol. 1991 Jul;65(7):3759–3769. doi: 10.1128/jvi.65.7.3759-3769.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cherry J. R., Johnson T. R., Dollard C., Shuster J. R., Denis C. L. Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADR1. Cell. 1989 Feb 10;56(3):409–419. doi: 10.1016/0092-8674(89)90244-4. [DOI] [PubMed] [Google Scholar]
  4. Cheung A. K. DNA nucleotide sequence analysis of the immediate-early gene of pseudorabies virus. Nucleic Acids Res. 1989 Jun 26;17(12):4637–4646. doi: 10.1093/nar/17.12.4637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  6. Courtney R. J., Benyesh-Melnick M. Isolation and characterization of a large molecular-weight polypeptide of herpes simplex virus type 1. Virology. 1974 Dec;62(2):539–551. doi: 10.1016/0042-6822(74)90414-0. [DOI] [PubMed] [Google Scholar]
  7. DeLuca N. A., Courtney M. A., Schaffer P. A. Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression. J Virol. 1984 Dec;52(3):767–776. doi: 10.1128/jvi.52.3.767-776.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeLuca N. A., McCarthy A. M., Schaffer P. A. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol. 1985 Nov;56(2):558–570. doi: 10.1128/jvi.56.2.558-570.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeLuca N. A., Schaffer P. A. Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4. Mol Cell Biol. 1985 Aug;5(8):1997–2008. doi: 10.1128/mcb.5.8.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeLuca N. A., Schaffer P. A. Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res. 1987 Jun 11;15(11):4491–4511. doi: 10.1093/nar/15.11.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLuca N. A., Schaffer P. A. Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4. J Virol. 1988 Mar;62(3):732–743. doi: 10.1128/jvi.62.3.732-743.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  13. Faber S. W., Wilcox K. W. Association of the herpes simplex virus regulatory protein ICP4 with specific nucleotide sequences in DNA. Nucleic Acids Res. 1986 Aug 11;14(15):6067–6083. doi: 10.1093/nar/14.15.6067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Faber S. W., Wilcox K. W. Characterization of a herpes simplex virus regulatory protein: aggregation and phosphorylation of a temperature-sensitive variant of ICP 4. Arch Virol. 1986;91(3-4):297–312. doi: 10.1007/BF01314289. [DOI] [PubMed] [Google Scholar]
  15. Flanagan W. M., Papavassiliou A. G., Rice M., Hecht L. B., Silverstein S., Wagner E. K. Analysis of the herpes simplex virus type 1 promoter controlling the expression of UL38, a true late gene involved in capsid assembly. J Virol. 1991 Feb;65(2):769–786. doi: 10.1128/jvi.65.2.769-786.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ge H., Roeder R. G. Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell. 1994 Aug 12;78(3):513–523. doi: 10.1016/0092-8674(94)90428-6. [DOI] [PubMed] [Google Scholar]
  17. Ghosh S., Baltimore D. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature. 1990 Apr 12;344(6267):678–682. doi: 10.1038/344678a0. [DOI] [PubMed] [Google Scholar]
  18. Gibson W., Roizman B. Proteins specified by herpes simplex virus. 8. Characterization and composition of multiple capsid forms of subtypes 1 and 2. J Virol. 1972 Nov;10(5):1044–1052. doi: 10.1128/jvi.10.5.1044-1052.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ginty D. D., Glowacka D., DeFranco C., Wagner J. A. Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities. J Biol Chem. 1991 Aug 15;266(23):15325–15333. [PubMed] [Google Scholar]
  20. Grundy F. J., Baumann R. P., O'Callaghan D. J. DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene. Virology. 1989 Sep;172(1):223–236. doi: 10.1016/0042-6822(89)90124-4. [DOI] [PubMed] [Google Scholar]
  21. Gu B., Kuddus R., DeLuca N. A. Repression of activator-mediated transcription by herpes simplex virus ICP4 via a mechanism involving interactions with the basal transcription factors TATA-binding protein and TFIIB. Mol Cell Biol. 1995 Jul;15(7):3618–3626. doi: 10.1128/mcb.15.7.3618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Honess R. W., Roizman B. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol. 1974 Jul;14(1):8–19. doi: 10.1128/jvi.14.1.8-19.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Honess R. W., Roizman B. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1276–1280. doi: 10.1073/pnas.72.4.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hope I. A., Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. doi: 10.1016/0092-8674(86)90070-x. [DOI] [PubMed] [Google Scholar]
  25. Imbalzano A. N., Shepard A. A., DeLuca N. A. Functional relevance of specific interactions between herpes simplex virus type 1 ICP4 and sequences from the promoter-regulatory domain of the viral thymidine kinase gene. J Virol. 1990 Jun;64(6):2620–2631. doi: 10.1128/jvi.64.6.2620-2631.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kattar-Cooley P., Wilcox K. W. Characterization of the DNA-binding properties of herpes simplex virus regulatory protein ICP4. J Virol. 1989 Feb;63(2):696–704. doi: 10.1128/jvi.63.2.696-704.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kemp B. E., Graves D. J., Benjamini E., Krebs E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem. 1977 Jul 25;252(14):4888–4894. [PubMed] [Google Scholar]
  29. Knipe D. M., Ruyechan W. T., Roizman B., Halliburton I. W. Molecular genetics of herpes simplex virus: demonstration of regions of obligatory and nonobligatory identity within diploid regions of the genome by sequence replacement and insertion. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3896–3900. doi: 10.1073/pnas.75.8.3896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kretzschmar M., Kaiser K., Lottspeich F., Meisterernst M. A novel mediator of class II gene transcription with homology to viral immediate-early transcriptional regulators. Cell. 1994 Aug 12;78(3):525–534. doi: 10.1016/0092-8674(94)90429-4. [DOI] [PubMed] [Google Scholar]
  31. Kristie T. M., Roizman B. Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes. Proc Natl Acad Sci U S A. 1986 May;83(10):3218–3222. doi: 10.1073/pnas.83.10.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kristie T. M., Roizman B. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4700–4704. doi: 10.1073/pnas.83.13.4700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Leib D. A., Coen D. M., Bogard C. L., Hicks K. A., Yager D. R., Knipe D. M., Tyler K. L., Schaffer P. A. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol. 1989 Feb;63(2):759–768. doi: 10.1128/jvi.63.2.759-768.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lüscher B., Christenson E., Litchfield D. W., Krebs E. G., Eisenman R. N. Myb DNA binding inhibited by phosphorylation at a site deleted during oncogenic activation. Nature. 1990 Apr 5;344(6266):517–522. doi: 10.1038/344517a0. [DOI] [PubMed] [Google Scholar]
  36. McGeoch D. J., Dolan A., Donald S., Brauer D. H. Complete DNA sequence of the short repeat region in the genome of herpes simplex virus type 1. Nucleic Acids Res. 1986 Feb 25;14(4):1727–1745. doi: 10.1093/nar/14.4.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Michael N., Roizman B. Binding of the herpes simplex virus major regulatory protein to viral DNA. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9808–9812. doi: 10.1073/pnas.86.24.9808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Michael N., Spector D., Mavromara-Nazos P., Kristie T. M., Roizman B. The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses. Science. 1988 Mar 25;239(4847):1531–1534. doi: 10.1126/science.2832940. [DOI] [PubMed] [Google Scholar]
  39. Moody A. J., Cooper C. E., Rich P. R. Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase. Biochim Biophys Acta. 1991 Aug 23;1059(2):189–207. doi: 10.1016/s0005-2728(05)80204-x. [DOI] [PubMed] [Google Scholar]
  40. Muller M. T. Binding of the herpes simplex virus immediate-early gene product ICP4 to its own transcription start site. J Virol. 1987 Mar;61(3):858–865. doi: 10.1128/jvi.61.3.858-865.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Papavassiliou A. G., Wilcox K. W., Silverstein S. J. The interaction of ICP4 with cell/infected-cell factors and its state of phosphorylation modulate differential recognition of leader sequences in herpes simplex virus DNA. EMBO J. 1991 Feb;10(2):397–406. doi: 10.1002/j.1460-2075.1991.tb07961.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Paterson T., Everett R. D. A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture. J Gen Virol. 1990 Aug;71(Pt 8):1775–1783. doi: 10.1099/0022-1317-71-8-1775. [DOI] [PubMed] [Google Scholar]
  43. Paterson T., Everett R. D. Mutational dissection of the HSV-1 immediate-early protein Vmw175 involved in transcriptional transactivation and repression. Virology. 1988 Sep;166(1):186–196. doi: 10.1016/0042-6822(88)90160-2. [DOI] [PubMed] [Google Scholar]
  44. Paterson T., Everett R. D. The regions of the herpes simplex virus type 1 immediate early protein Vmw175 required for site specific DNA binding closely correspond to those involved in transcriptional regulation. Nucleic Acids Res. 1988 Dec 9;16(23):11005–11025. doi: 10.1093/nar/16.23.11005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pearson R. B., Kemp B. E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. doi: 10.1016/0076-6879(91)00127-i. [DOI] [PubMed] [Google Scholar]
  46. Pereira L., Wolff M. H., Fenwick M., Roizman B. Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells. Virology. 1977 Apr;77(2):733–749. doi: 10.1016/0042-6822(77)90495-0. [DOI] [PubMed] [Google Scholar]
  47. Preston C. M. Abnormal properties of an immediate early polypeptide in cells infected with the herpes simplex virus type 1 mutant tsK. J Virol. 1979 Nov;32(2):357–369. doi: 10.1128/jvi.32.2.357-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Preston C. M. Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK. J Virol. 1979 Jan;29(1):275–284. doi: 10.1128/jvi.29.1.275-284.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Preston C. M., Notarianni E. L. Poly(ADP-ribosyl)ation of a herpes simplex virus immediate early polypeptide. Virology. 1983 Dec;131(2):492–501. doi: 10.1016/0042-6822(83)90515-9. [DOI] [PubMed] [Google Scholar]
  50. Preston V. G., Davison A. J., Marsden H. S., Timbury M. C., Subak-Sharpe J. H., Wilkie N. M. Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides. J Virol. 1978 Nov;28(2):499–517. doi: 10.1128/jvi.28.2.499-517.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  52. Reed J., Kinzel V., Kemp B. E., Cheng H. C., Walsh D. A. Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase. Biochemistry. 1985 Jun 4;24(12):2967–2973. doi: 10.1021/bi00333a024. [DOI] [PubMed] [Google Scholar]
  53. Riabowol K. T., Fink J. S., Gilman M. Z., Walsh D. A., Goodman R. H., Feramisco J. R. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. Nature. 1988 Nov 3;336(6194):83–86. doi: 10.1038/336083a0. [DOI] [PubMed] [Google Scholar]
  54. Roskoski R., Jr Assays of protein kinase. Methods Enzymol. 1983;99:3–6. doi: 10.1016/0076-6879(83)99034-1. [DOI] [PubMed] [Google Scholar]
  55. Shepard A. A., DeLuca N. A. A second-site revertant of a defective herpes simplex virus ICP4 protein with restored regulatory activities and impaired DNA-binding properties. J Virol. 1991 Feb;65(2):787–795. doi: 10.1128/jvi.65.2.787-795.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Shepard A. A., DeLuca N. A. Activities of heterodimers composed of DNA-binding- and transactivation-deficient subunits of the herpes simplex virus regulatory protein ICP4. J Virol. 1991 Jan;65(1):299–307. doi: 10.1128/jvi.65.1.299-307.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shepard A. A., Imbalzano A. N., DeLuca N. A. Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes simplex virus transcriptional regulatory protein ICP4. J Virol. 1989 Sep;63(9):3714–3728. doi: 10.1128/jvi.63.9.3714-3728.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Smith C. A., Bates P., Rivera-Gonzalez R., Gu B., DeLuca N. A. ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB. J Virol. 1993 Aug;67(8):4676–4687. doi: 10.1128/jvi.67.8.4676-4687.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vlcek C., Paces V., Schwyzer M. Nucleotide sequence of the pseudorabies virus immediate early gene, encoding a strong transactivator protein. Virus Genes. 1989 Aug;2(4):335–346. doi: 10.1007/BF00684041. [DOI] [PubMed] [Google Scholar]
  60. Watson R. J., Clements J. B. A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature. 1980 May 29;285(5763):329–330. doi: 10.1038/285329a0. [DOI] [PubMed] [Google Scholar]
  61. Whitby A. J., Blyth W. A., Hill T. J. The effect of DNA hypomethylating agents on the reactivation of herpes simplex virus from latently infected mouse ganglia in vitro. Brief report. Arch Virol. 1987;97(1-2):137–144. doi: 10.1007/BF01310742. [DOI] [PubMed] [Google Scholar]
  62. Wilcox K. W., Kohn A., Sklyanskaya E., Roizman B. Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. J Virol. 1980 Jan;33(1):167–182. doi: 10.1128/jvi.33.1.167-182.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Xia K., DeLuca N. A., Knipe D. M. Analysis of phosphorylation sites of herpes simplex virus type 1 ICP4. J Virol. 1996 Feb;70(2):1061–1071. doi: 10.1128/jvi.70.2.1061-1071.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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