Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Mar 15;90(6):2286–2290. doi: 10.1073/pnas.90.6.2286

Repression of the herpes simplex virus 1 alpha 4 gene by its gene product occurs within the context of the viral genome and is associated with all three identified cognate sites.

N Michael 1, B Roizman 1
PMCID: PMC46071  PMID: 8384719

Abstract

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha 4-1 proximal, alpha 4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha 4-2) of the alpha 4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha 22 gene and 3 hr later with the test viruses were tested in RNase protection assay for amounts of the chimeric gene RNA relative to amounts of alpha 22 gene RNA. We report the following: (i) Mutation of the alpha 4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha 4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha 4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha 4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha 4 mRNA relative to that of alpha 22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.

Full text

PDF
2290

Images in this article

2288Fig. 3
on p.2288
2288Fig. 4
on p.2288

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. 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]
  3. DiDonato J. A., Muller M. T. DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element. J Virol. 1989 Sep;63(9):3737–3747. doi: 10.1128/jvi.63.9.3737-3747.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elshiekh N. A., Harris-Hamilton E., Bachenheimer S. L. Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis. J Virol. 1991 Dec;65(12):6430–6437. doi: 10.1128/jvi.65.12.6430-6437.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Everett R. D., Elliott M., Hope G., Orr A. Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site. Nucleic Acids Res. 1991 Sep 25;19(18):4901–4908. doi: 10.1093/nar/19.18.4901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Everett R. D., Orr A. The Vmw175 binding site in the IE-1 promoter has no apparent role in the expression of Vmw110 during herpes simplex virus type 1 infection. Virology. 1991 Feb;180(2):509–517. doi: 10.1016/0042-6822(91)90064-i. [DOI] [PubMed] [Google Scholar]
  7. Faber S. W., Wilcox K. W. Association of herpes simplex virus regulatory protein ICP4 with sequences spanning the ICP4 gene transcription initiation site. Nucleic Acids Res. 1988 Jan 25;16(2):555–570. doi: 10.1093/nar/16.2.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Fenwick M., Morse L. S., Roizman B. Anatomy of herpes simplex virus DNA. XI. Apparent clustering of functions effecting rapid inhibition of host DNA and protein synthesis. J Virol. 1979 Feb;29(2):825–827. doi: 10.1128/jvi.29.2.825-827.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fenwick M., Roizman B. Regulation of herpesvirus macromolecular synthesis. VI. Synthesis and modification of viral polypeptides in enucleated cells. J Virol. 1977 Jun;22(3):720–725. doi: 10.1128/jvi.22.3.720-725.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  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. Honess R. W., Roizman B. Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J Virol. 1973 Dec;12(6):1347–1365. doi: 10.1128/jvi.12.6.1347-1365.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Kristie T. M., Roizman B. Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4065–4069. doi: 10.1073/pnas.81.13.4065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mackem S., Roizman B. Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes. J Virol. 1982 Dec;44(3):939–949. doi: 10.1128/jvi.44.3.939-949.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. O'Hare P., Hayward G. S. Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded trans-acting factors. J Virol. 1987 Jan;61(1):190–199. doi: 10.1128/jvi.61.1.190-199.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Panning B., Smiley J. R. Regulation of cellular genes transduced by herpes simplex virus. J Virol. 1989 May;63(5):1929–1937. doi: 10.1128/jvi.63.5.1929-1937.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Papavassiliou A. G., Silverstein S. J. Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene. J Biol Chem. 1990 Jan 25;265(3):1648–1657. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Paterson T., Preston V. G., Everett R. D. A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo. J Gen Virol. 1990 Apr;71(Pt 4):851–861. doi: 10.1099/0022-1317-71-4-851. [DOI] [PubMed] [Google Scholar]
  31. Post L. E., Roizman B. A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth. Cell. 1981 Jul;25(1):227–232. doi: 10.1016/0092-8674(81)90247-6. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Roizman B., Kristie T., McKnight J. L., Michael N., Mavromara-Nazos P., Spector D. The trans-activation of herpes simplex virus gene expression: comparison of two factors and their cis sites. Biochimie. 1988 Aug;70(8):1031–1043. doi: 10.1016/0300-9084(88)90266-0. [DOI] [PubMed] [Google Scholar]
  34. Romanelli M. G., Mavromara-Nazos P., Spector D., Roizman B. Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene. J Virol. 1992 Aug;66(8):4855–4863. doi: 10.1128/jvi.66.8.4855-4863.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sears A. E., McGwire B. S., Roizman B. Infection of polarized MDCK cells with herpes simplex virus 1: two asymmetrically distributed cell receptors interact with different viral proteins. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5087–5091. doi: 10.1073/pnas.88.12.5087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Silver S., Roizman B. gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes. Mol Cell Biol. 1985 Mar;5(3):518–528. doi: 10.1128/mcb.5.3.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smiley J. R., Johnson D. C., Pizer L. I., Everett R. D. The ICP4 binding sites in the herpes simplex virus type 1 glycoprotein D (gD) promoter are not essential for efficient gD transcription during virus infection. J Virol. 1992 Feb;66(2):623–631. doi: 10.1128/jvi.66.2.623-631.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smiley J. R., Smibert C., Everett R. D. Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts. J Virol. 1987 Aug;61(8):2368–2377. doi: 10.1128/jvi.61.8.2368-2377.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Spector D., Purves F., Roizman B. Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5268–5272. doi: 10.1073/pnas.87.14.5268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tedder D. G., Everett R. D., Wilcox K. W., Beard P., Pizer L. I. ICP4-binding sites in the promoter and coding regions of the herpes simplex virus gD gene contribute to activation of in vitro transcription by ICP4. J Virol. 1989 Jun;63(6):2510–2520. doi: 10.1128/jvi.63.6.2510-2520.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES