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. 1993 Feb;67(2):876–885. doi: 10.1128/jvi.67.2.876-885.1993

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

M Gao 1, D M Knipe 1
PMCID: PMC237441  PMID: 8380469

Abstract

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.

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Selected References

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

  1. Bayliss G. J., Marsden H. S., Hay J. Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure. Virology. 1975 Nov;68(1):124–134. doi: 10.1016/0042-6822(75)90154-3. [DOI] [PubMed] [Google Scholar]
  2. Capson T. L., Benkovic S. J., Nossal N. G. Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template. Cell. 1991 Apr 19;65(2):249–258. doi: 10.1016/0092-8674(91)90159-v. [DOI] [PubMed] [Google Scholar]
  3. Challberg M. D. A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9094–9098. doi: 10.1073/pnas.83.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conley A. J., Knipe D. M., Jones P. C., Roizman B. Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides. J Virol. 1981 Jan;37(1):191–206. doi: 10.1128/jvi.37.1.191-206.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Gao M., Bouchey J., Curtin K., Knipe D. M. Genetic identification of a portion of the herpes simplex virus ICP8 protein required for DNA-binding. Virology. 1988 Apr;163(2):319–329. doi: 10.1016/0042-6822(88)90272-3. [DOI] [PubMed] [Google Scholar]
  7. Gao M., Knipe D. M. Distal protein sequences can affect the function of a nuclear localization signal. Mol Cell Biol. 1992 Mar;12(3):1330–1339. doi: 10.1128/mcb.12.3.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gao M., Knipe D. M. Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein. J Virol. 1989 Dec;63(12):5258–5267. doi: 10.1128/jvi.63.12.5258-5267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao M., Knipe D. M. Potential role for herpes simplex virus ICP8 DNA replication protein in stimulation of late gene expression. J Virol. 1991 May;65(5):2666–2675. doi: 10.1128/jvi.65.5.2666-2675.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  12. Knipe D. M., Quinlan M. P., Spang A. E. Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1. J Virol. 1982 Nov;44(2):736–741. doi: 10.1128/jvi.44.2.736-741.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Knipe D. M., Ruyechan W. T., Roizman B. Molecular genetics of herpes simplex virus. III. Fine mapping of a genetic locus determining resistance to phosphonoacetate by two methods of marker transfer. J Virol. 1979 Feb;29(2):698–704. doi: 10.1128/jvi.29.2.698-704.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lee C. K., Knipe D. M. An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8. J Virol. 1985 Jun;54(3):731–738. doi: 10.1128/jvi.54.3.731-738.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee C. K., Knipe D. M. Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1. J Virol. 1983 Jun;46(3):909–919. doi: 10.1128/jvi.46.3.909-919.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leinbach S. S., Heath L. S. A carboxyl-terminal peptide of the DNA-binding protein ICP8 of herpes simplex virus contains a single-stranded DNA-binding site. Virology. 1988 Sep;166(1):10–16. doi: 10.1016/0042-6822(88)90140-7. [DOI] [PubMed] [Google Scholar]
  18. Littler E., Purifoy D., Minson A., Powell K. L. Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein. J Gen Virol. 1983 May;64(Pt 5):983–995. doi: 10.1099/0022-1317-64-5-983. [DOI] [PubMed] [Google Scholar]
  19. O'Donnell M. E., Elias P., Funnell B. E., Lehman I. R. Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1. J Biol Chem. 1987 Mar 25;262(9):4260–4266. [PubMed] [Google Scholar]
  20. Orberg P. K., Schaffer P. A. Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus. J Virol. 1987 Apr;61(4):1136–1146. doi: 10.1128/jvi.61.4.1136-1146.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Powell K. L., Littler E., Purifoy D. J. Nonstructural proteins of herpes simplex virus. II. Major virus-specific DNa-binding protein. J Virol. 1981 Sep;39(3):894–902. doi: 10.1128/jvi.39.3.894-902.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Powell K. L., Purifoy D. J. DNA-binding proteins of cells infected by herpes simplex virus type 1 and type 2. Intervirology. 1976;7(4-5):225–239. doi: 10.1159/000149955. [DOI] [PubMed] [Google Scholar]
  23. Quinlan M. P., Chen L. B., Knipe D. M. The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication. Cell. 1984 Apr;36(4):857–868. doi: 10.1016/0092-8674(84)90035-7. [DOI] [PubMed] [Google Scholar]
  24. Quinlan M. P., Knipe D. M. A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid. J Virol. 1985 May;54(2):619–622. doi: 10.1128/jvi.54.2.619-622.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quinlan M. P., Knipe D. M. Nuclear localization of herpesvirus proteins: potential role for the cellular framework. Mol Cell Biol. 1983 Mar;3(3):315–324. doi: 10.1128/mcb.3.3.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rice S. A., Knipe D. M. Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27. J Virol. 1990 Apr;64(4):1704–1715. doi: 10.1128/jvi.64.4.1704-1715.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rose D. S., Shriver K., Latchman D. S., LaThangue N. B. A filamentous distribution for the herpes simplex virus type 2-encoded major DNA-binding protein. J Gen Virol. 1986 Jul;67(Pt 7):1315–1325. doi: 10.1099/0022-1317-67-7-1315. [DOI] [PubMed] [Google Scholar]
  28. Ruyechan W. T., Chytil A., Fisher C. M. In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein. J Virol. 1986 Jul;59(1):31–36. doi: 10.1128/jvi.59.1.31-36.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ruyechan W. T. The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration. J Virol. 1983 May;46(2):661–666. doi: 10.1128/jvi.46.2.661-666.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  33. Wang Y. S., Hall J. D. Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). J Virol. 1990 May;64(5):2082–2089. doi: 10.1128/jvi.64.5.2082-2089.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weller S. K., Lee K. J., Sabourin D. J., Schaffer P. A. Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein. J Virol. 1983 Jan;45(1):354–366. doi: 10.1128/jvi.45.1.354-366.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wu C. A., Nelson N. J., McGeoch D. J., Challberg M. D. Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. J Virol. 1988 Feb;62(2):435–443. doi: 10.1128/jvi.62.2.435-443.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Bruyn Kops A., Knipe D. M. Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein. Cell. 1988 Dec 2;55(5):857–868. doi: 10.1016/0092-8674(88)90141-9. [DOI] [PubMed] [Google Scholar]

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