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. 1994 Feb 11;22(3):270–278. doi: 10.1093/nar/22.3.270

Mutation of a single lysine residue severely impairs the DNA recognition and regulatory functions of the VZV gene 62 transactivator protein.

J K Tyler 1, K E Allen 1, R D Everett 1
PMCID: PMC523576  PMID: 7907417

Abstract

The product of varicella-zoster virus gene 62 (VZV 140k) is a potent transactivator protein. We have identified a region within the DNA binding domain of VZV 140k that shows a striking similarity to the DNA recognition helix of the homeodomain, with an especially highly conserved quartet of residues, WLQN. The 140k protein has functional counterparts within the other alphaherpesviruses, which include the major transcriptional regulatory protein of HSV-1, (ICP4), and the WLQN region is highly conserved among the members of this family of viral transactivators. Substitution of VZV 140k residue lysine 548, just adjacent to the WLQN region, drastically reduces the DNA binding activity of the 140k DNA binding domain and the intact 140k mutant protein fails to activate gene expression. Substitutions of two other VZV 140k residues in this conserved WLQN region result in alterations to the DNA binding interaction and reduced transactivation activities. All three mutations act at the level of DNA recognition, as they have no apparent effect on the dimerization state, solubility or efficiency of expression of the mutant peptides.

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Selected References

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  1. Affolter M., Percival-Smith A., Müller M., Leupin W., Gehring W. J. DNA binding properties of the purified Antennapedia homeodomain. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4093–4097. doi: 10.1073/pnas.87.11.4093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arthur A. K., Höss A., Fanning E. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J Virol. 1988 Jun;62(6):1999–2006. doi: 10.1128/jvi.62.6.1999-2006.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Betz J. L., Wydoski S. G. Functional interaction of varicella zoster virus gene 62 protein with the DNA sequence bound by herpes simplex virus ICP4 protein. Virology. 1993 Aug;195(2):793–797. doi: 10.1006/viro.1993.1432. [DOI] [PubMed] [Google Scholar]
  4. Brennan R. G. The winged-helix DNA-binding motif: another helix-turn-helix takeoff. Cell. 1993 Sep 10;74(5):773–776. doi: 10.1016/0092-8674(93)90456-z. [DOI] [PubMed] [Google Scholar]
  5. Ceska T. A., Lamers M., Monaci P., Nicosia A., Cortese R., Suck D. The X-ray structure of an atypical homeodomain present in the rat liver transcription factor LFB1/HNF1 and implications for DNA binding. EMBO J. 1993 May;12(5):1805–1810. doi: 10.2210/pdb1lfb/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Clark K. L., Halay E. D., Lai E., Burley S. K. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 1993 Jul 29;364(6436):412–420. doi: 10.1038/364412a0. [DOI] [PubMed] [Google Scholar]
  8. Clarke N. D., Beamer L. J., Goldberg H. R., Berkower C., Pabo C. O. The DNA binding arm of lambda repressor: critical contacts from a flexible region. Science. 1991 Oct 11;254(5029):267–270. doi: 10.1126/science.254.5029.267. [DOI] [PubMed] [Google Scholar]
  9. Corsaro C. M., Pearson M. L. Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells. Somatic Cell Genet. 1981 Sep;7(5):603–616. doi: 10.1007/BF01549662. [DOI] [PubMed] [Google Scholar]
  10. Davison A. J., Scott J. E. The complete DNA sequence of varicella-zoster virus. J Gen Virol. 1986 Sep;67(Pt 9):1759–1816. doi: 10.1099/0022-1317-67-9-1759. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. DiDonato J. A., Spitzner J. R., Muller M. T. A predictive model for DNA recognition by the herpes simplex virus protein ICP4. J Mol Biol. 1991 Jun 5;219(3):451–470. doi: 10.1016/0022-2836(91)90186-a. [DOI] [PubMed] [Google Scholar]
  13. Disney G. H., Everett R. D. A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame. J Gen Virol. 1990 Nov;71(Pt 11):2681–2689. doi: 10.1099/0022-1317-71-11-2681. [DOI] [PubMed] [Google Scholar]
  14. Disney G. H., McKee T. A., Preston C. M., Everett R. D. The product of varicella-zoster virus gene 62 autoregulates its own promoter. J Gen Virol. 1990 Dec;71(Pt 12):2999–3003. doi: 10.1099/0022-1317-71-12-2999. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Everett R. D. Analysis of the functional domains of herpes simplex virus type 1 immediate-early polypeptide Vmw110. J Mol Biol. 1988 Jul 5;202(1):87–96. doi: 10.1016/0022-2836(88)90521-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Everett R. D., Paterson T., Elliott M. The major transcriptional regulatory protein of herpes simplex virus type 1 includes a protease resistant DNA binding domain. Nucleic Acids Res. 1990 Aug 11;18(15):4579–4585. doi: 10.1093/nar/18.15.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Everett R. D. The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans. J Gen Virol. 1986 Nov;67(Pt 11):2507–2513. doi: 10.1099/0022-1317-67-11-2507. [DOI] [PubMed] [Google Scholar]
  20. Everett R. D. The regulation of transcription of viral and cellular genes by herpesvirus immediate-early gene products (review). Anticancer Res. 1987 Jul-Aug;7(4A):589–604. [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Felser J. M., Kinchington P. R., Inchauspe G., Straus S. E., Ostrove J. M. Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1. J Virol. 1988 Jun;62(6):2076–2082. doi: 10.1128/jvi.62.6.2076-2082.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fortini M. E., Lai Z. C., Rubin G. M. The Drosophila zfh-1 and zfh-2 genes encode novel proteins containing both zinc-finger and homeodomain motifs. Mech Dev. 1991 Jun;34(2-3):113–122. doi: 10.1016/0925-4773(91)90048-b. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Hanes S. D., Brent R. A genetic model for interaction of the homeodomain recognition helix with DNA. Science. 1991 Jan 25;251(4992):426–430. doi: 10.1126/science.1671176. [DOI] [PubMed] [Google Scholar]
  30. Hanes S. D., Brent R. DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell. 1989 Jun 30;57(7):1275–1283. doi: 10.1016/0092-8674(89)90063-9. [DOI] [PubMed] [Google Scholar]
  31. Harrison S. C. A structural taxonomy of DNA-binding domains. Nature. 1991 Oct 24;353(6346):715–719. doi: 10.1038/353715a0. [DOI] [PubMed] [Google Scholar]
  32. Herr W., Sturm R. A., Clerc R. G., Corcoran L. M., Baltimore D., Sharp P. A., Ingraham H. A., Rosenfeld M. G., Finney M., Ruvkun G. The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes Dev. 1988 Dec;2(12A):1513–1516. doi: 10.1101/gad.2.12a.1513. [DOI] [PubMed] [Google Scholar]
  33. Inchauspe G., Nagpal S., Ostrove J. M. Mapping of two varicella-zoster virus-encoded genes that activate the expression of viral early and late genes. Virology. 1989 Dec;173(2):700–709. doi: 10.1016/0042-6822(89)90583-7. [DOI] [PubMed] [Google Scholar]
  34. Inchauspe G., Ostrove J. M. Differential regulation by varicella-zoster virus (VZV) and herpes simplex virus type-1 trans-activating genes. Virology. 1989 Dec;173(2):710–714. doi: 10.1016/0042-6822(89)90584-9. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Kissinger C. R., Liu B. S., Martin-Blanco E., Kornberg T. B., Pabo C. O. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions. Cell. 1990 Nov 2;63(3):579–590. doi: 10.1016/0092-8674(90)90453-l. [DOI] [PubMed] [Google Scholar]
  37. Laughon A. DNA binding specificity of homeodomains. Biochemistry. 1991 Dec 3;30(48):11357–11367. doi: 10.1021/bi00112a001. [DOI] [PubMed] [Google Scholar]
  38. MACPHERSON I., STOKER M. Polyoma transformation of hamster cell clones--an investigation of genetic factors affecting cell competence. Virology. 1962 Feb;16:147–151. doi: 10.1016/0042-6822(62)90290-8. [DOI] [PubMed] [Google Scholar]
  39. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. McKee T. A., Disney G. H., Everett R. D., Preston C. M. Control of expression of the varicella-zoster virus major immediate early gene. J Gen Virol. 1990 Apr;71(Pt 4):897–906. doi: 10.1099/0022-1317-71-4-897. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Otting G., Qian Y. Q., Billeter M., Müller M., Affolter M., Gehring W. J., Wüthrich K. Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution. EMBO J. 1990 Oct;9(10):3085–3092. doi: 10.1002/j.1460-2075.1990.tb07505.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Perera L. P., Mosca J. D., Ruyechan W. T., Hay J. Regulation of varicella-zoster virus gene expression in human T lymphocytes. J Virol. 1992 Sep;66(9):5298–5304. doi: 10.1128/jvi.66.9.5298-5304.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Perera L. P., Mosca J. D., Sadeghi-Zadeh M., Ruyechan W. T., Hay J. The varicella-zoster virus immediate early protein, IE62, can positively regulate its cognate promoter. Virology. 1992 Nov;191(1):346–354. doi: 10.1016/0042-6822(92)90197-w. [DOI] [PubMed] [Google Scholar]
  50. Pizer L. I., Everett R. D., Tedder D. G., Elliott M., Litman B. Nucleotides within both proximal and distal parts of the consensus sequence are important for specific DNA recognition by the herpes simplex virus regulatory protein ICP4. Nucleic Acids Res. 1991 Feb 11;19(3):477–483. doi: 10.1093/nar/19.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Post L. E., Mackem S., Roizman B. Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell. 1981 May;24(2):555–565. doi: 10.1016/0092-8674(81)90346-9. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Qian Y. Q., Billeter M., Otting G., Müller M., Gehring W. J., Wüthrich K. The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors. Cell. 1989 Nov 3;59(3):573–580. doi: 10.1016/0092-8674(89)90040-8. [DOI] [PubMed] [Google Scholar]
  54. Ruvkun G., Finney M. Regulation of transcription and cell identity by POU domain proteins. Cell. 1991 Feb 8;64(3):475–478. doi: 10.1016/0092-8674(91)90227-p. [DOI] [PubMed] [Google Scholar]
  55. Sauer R. T., Smith D. L., Johnson A. D. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. doi: 10.1101/gad.2.7.807. [DOI] [PubMed] [Google Scholar]
  56. Scott M. P., Tamkun J. W., Hartzell G. W., 3rd The structure and function of the homeodomain. Biochim Biophys Acta. 1989 Jul 28;989(1):25–48. doi: 10.1016/0304-419x(89)90033-4. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. Stow N. D. Herpes simplex virus type 1 origin-dependent DNA replication in insect cells using recombinant baculoviruses. J Gen Virol. 1992 Feb;73(Pt 2):313–321. doi: 10.1099/0022-1317-73-2-313. [DOI] [PubMed] [Google Scholar]
  60. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  61. Suzuki M. Common features in DNA recognition helices of eukaryotic transcription factors. EMBO J. 1993 Aug;12(8):3221–3226. doi: 10.1002/j.1460-2075.1993.tb05991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Telford E. A., Watson M. S., McBride K., Davison A. J. The DNA sequence of equine herpesvirus-1. Virology. 1992 Jul;189(1):304–316. doi: 10.1016/0042-6822(92)90706-u. [DOI] [PubMed] [Google Scholar]
  63. Treisman J., Gönczy P., Vashishtha M., Harris E., Desplan C. A single amino acid can determine the DNA binding specificity of homeodomain proteins. Cell. 1989 Nov 3;59(3):553–562. doi: 10.1016/0092-8674(89)90038-x. [DOI] [PubMed] [Google Scholar]
  64. Treisman J., Harris E., Desplan C. The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev. 1991 Apr;5(4):594–604. doi: 10.1101/gad.5.4.594. [DOI] [PubMed] [Google Scholar]
  65. Tyler J. K., Everett R. D. The DNA binding domain of the varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1. Nucleic Acids Res. 1993 Feb 11;21(3):513–522. doi: 10.1093/nar/21.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wolberger C., Vershon A. K., Liu B., Johnson A. D., Pabo C. O. Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions. Cell. 1991 Nov 1;67(3):517–528. doi: 10.1016/0092-8674(91)90526-5. [DOI] [PubMed] [Google Scholar]
  67. 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]
  68. 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]

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