Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Feb;70(2):1061–1071. doi: 10.1128/jvi.70.2.1061-1071.1996

Analysis of phosphorylation sites of herpes simplex virus type 1 ICP4.

K Xia 1, N A DeLuca 1, D M Knipe 1
PMCID: PMC189912  PMID: 8551564

Abstract

The herpes simplex virus ICP4 protein is required for induction of early and late viral gene transcription as well as for repression of expression of its own gene and several other viral genes. Several electrophoretic forms of ICP4 have been observed, and phosphorylation is thought to contribute to this heterogeneity and possibly to the multiple functions of ICP4. To define the complexity of the site(s) of phosphorylation of ICP4 and to initiate mapping of this site(s), we have performed two-dimensional phosphopeptide mapping of wild-type and mutant forms of ICP4 labeled in infected cells or in vitro. Wild-type ICP4 labeled in infected cells shows a complex pattern of phosphopeptides, and smaller mutant forms of ICP4 show progressively fewer phosphopeptides, arguing that multiple sites on ICP4 are phosphorylated. The serine-rich region of ICP4, residues 175 to 198, was shown to be a site for phosphorylation. Furthermore, the serine-rich region itself or the phosphorylation of this region increases phosphorylation of all phosphopeptides. A mutant ICP4 molecule lacking the serine-rich region showed low levels of phosphorylation by protein kinase A or protein kinase C in vitro. These results suggest that there may be a sequential phosphorylation of ICP4, with phosphorylation of the serine-rich region stimulating phosphorylation of the rest of the molecule. In addition, purified ICP4 showed an associated kinase activity or an autophosphorylation activity with properties different from those of protein kinase A or protein kinase C.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  4. Chapman C. J., Harris J. D., Hardwicke M. A., Sandri-Goldin R. M., Collins M. K., Latchman D. S. Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27. Virology. 1992 Feb;186(2):573–578. doi: 10.1016/0042-6822(92)90023-i. [DOI] [PubMed] [Google Scholar]
  5. Collett M. S., Erikson R. L. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2021–2024. doi: 10.1073/pnas.75.4.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Dixon R. A., Schaffer P. A. Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175. J Virol. 1980 Oct;36(1):189–203. doi: 10.1128/jvi.36.1.189-203.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Everett R. D. Trans activation of transcription by herpes virus products: requirement for two HSV-1 immediate-early polypeptides for maximum activity. EMBO J. 1984 Dec 20;3(13):3135–3141. doi: 10.1002/j.1460-2075.1984.tb02270.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gelman I. H., Silverstein S. Identification of immediate early genes from herpes simplex virus that transactivate the virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5265–5269. doi: 10.1073/pnas.82.16.5265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Godowski P. J., Knipe D. M. Transcriptional control of herpesvirus gene expression: gene functions required for positive and negative regulation. Proc Natl Acad Sci U S A. 1986 Jan;83(2):256–260. doi: 10.1073/pnas.83.2.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gu B., DeLuca N. Requirements for activation of the herpes simplex virus glycoprotein C promoter in vitro by the viral regulatory protein ICP4. J Virol. 1994 Dec;68(12):7953–7965. doi: 10.1128/jvi.68.12.7953-7965.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
  16. Hardy W. R., Sandri-Goldin R. M. Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J Virol. 1994 Dec;68(12):7790–7799. doi: 10.1128/jvi.68.12.7790-7799.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Knipe D. M., Spang A. E. Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus. J Virol. 1982 Jul;43(1):314–324. doi: 10.1128/jvi.43.1.314-324.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kramer M. F., Coen D. M. Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus. J Virol. 1995 Mar;69(3):1389–1399. doi: 10.1128/jvi.69.3.1389-1399.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Leader D. P., Katan M. Viral aspects of protein phosphorylation. J Gen Virol. 1988 Jul;69(Pt 7):1441–1464. doi: 10.1099/0022-1317-69-7-1441. [DOI] [PubMed] [Google Scholar]
  25. McCarthy A. M., McMahan L., Schaffer P. A. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J Virol. 1989 Jan;63(1):18–27. doi: 10.1128/jvi.63.1.18-27.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McLauchlan J., Phelan A., Loney C., Sandri-Goldin R. M., Clements J. B. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. J Virol. 1992 Dec;66(12):6939–6945. doi: 10.1128/jvi.66.12.6939-6945.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McMahan L., Schaffer P. A. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection. J Virol. 1990 Jul;64(7):3471–3485. doi: 10.1128/jvi.64.7.3471-3485.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Mullen M. A., Ciufo D. M., Hayward G. S. Mapping of intracellular localization domains and evidence for colocalization interactions between the IE110 and IE175 nuclear transactivator proteins of herpes simplex virus. J Virol. 1994 May;68(5):3250–3266. doi: 10.1128/jvi.68.5.3250-3266.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. O'Hare P., Hayward G. S. Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters. J Virol. 1985 Mar;53(3):751–760. doi: 10.1128/jvi.53.3.751-760.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Hare P., Hayward G. S. Three trans-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation. J Virol. 1985 Dec;56(3):723–733. doi: 10.1128/jvi.56.3.723-733.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Quinlan M. P., Knipe D. M. Stimulation of expression of a herpes simplex virus DNA-binding protein by two viral functions. Mol Cell Biol. 1985 May;5(5):957–963. doi: 10.1128/mcb.5.5.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rice S. A., Knipe D. M. Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27. J Virol. 1988 Oct;62(10):3814–3823. doi: 10.1128/jvi.62.10.3814-3823.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rice S. A., Su L. S., Knipe D. M. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J Virol. 1989 Aug;63(8):3399–3407. doi: 10.1128/jvi.63.8.3399-3407.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ridley R. G., Patel H. V., Gerber G. E., Morton R. C., Freeman K. B. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence. Nucleic Acids Res. 1986 May 27;14(10):4025–4035. doi: 10.1093/nar/14.10.4025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Roberts M. S., Boundy A., O'Hare P., Pizzorno M. C., Ciufo D. M., Hayward G. S. Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein. J Virol. 1988 Nov;62(11):4307–4320. doi: 10.1128/jvi.62.11.4307-4320.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sacks W. R., Greene C. C., Aschman D. P., Schaffer P. A. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol. 1985 Sep;55(3):796–805. doi: 10.1128/jvi.55.3.796-805.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Samaniego L. A., Webb A. L., DeLuca N. A. Functional interactions between herpes simplex virus immediate-early proteins during infection: gene expression as a consequence of ICP27 and different domains of ICP4. J Virol. 1995 Sep;69(9):5705–5715. doi: 10.1128/jvi.69.9.5705-5715.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sandri-Goldin R. M., Mendoza G. E. A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Genes Dev. 1992 May;6(5):848–863. doi: 10.1101/gad.6.5.848. [DOI] [PubMed] [Google Scholar]
  47. Schlessinger J. Allosteric regulation of the epidermal growth factor receptor kinase. J Cell Biol. 1986 Dec;103(6 Pt 1):2067–2072. doi: 10.1083/jcb.103.6.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Shepard A. A., Tolentino P., DeLuca N. A. trans-dominant inhibition of herpes simplex virus transcriptional regulatory protein ICP4 by heterodimer formation. J Virol. 1990 Aug;64(8):3916–3926. doi: 10.1128/jvi.64.8.3916-3926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Showalter S. D., Zweig M., Hampar B. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4. Infect Immun. 1981 Dec;34(3):684–692. doi: 10.1128/iai.34.3.684-692.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Su L., Knipe D. M. Herpes simplex virus alpha protein ICP27 can inhibit or augment viral gene transactivation. Virology. 1989 Jun;170(2):496–504. doi: 10.1016/0042-6822(89)90441-8. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Watson R. J., Clements J. B. Characterization of transcription-deficient temperature-sensitive mutants of herpes simplex virus type 1. Virology. 1978 Dec;91(2):364–379. doi: 10.1016/0042-6822(78)90384-7. [DOI] [PubMed] [Google Scholar]
  55. Wettenhall R. E., Morgan F. J. Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of the major phosphorylation sites for cAMP-dependent protein kinases. J Biol Chem. 1984 Feb 25;259(4):2084–2091. [PubMed] [Google Scholar]
  56. 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]
  57. Wu C. L., Wilcox K. W. Codons 262 to 490 from the herpes simplex virus ICP4 gene are sufficient to encode a sequence-specific DNA binding protein. Nucleic Acids Res. 1990 Feb 11;18(3):531–538. doi: 10.1093/nar/18.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wu C. L., Wilcox K. W. The conserved DNA-binding domains encoded by the herpes simplex virus type 1 ICP4, pseudorabies virus IE180, and varicella-zoster virus ORF62 genes recognize similar sites in the corresponding promoters. J Virol. 1991 Mar;65(3):1149–1159. doi: 10.1128/jvi.65.3.1149-1159.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Xia K., Knipe D. M., DeLuca N. A. Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication. J Virol. 1996 Feb;70(2):1050–1060. doi: 10.1128/jvi.70.2.1050-1060.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES