Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Feb;68(2):823–833. doi: 10.1128/jvi.68.2.823-833.1994

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

S A Rice 1, V Lam 1
PMCID: PMC236519  PMID: 8289386

Abstract

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.

Full text

PDF
823

Images in this article

828Image
on p.828
828Image
on p.828
829Image
on p.829
830Image
on p.830
830Image
on p.830

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. Albrecht J. C., Nicholas J., Biller D., Cameron K. R., Biesinger B., Newman C., Wittmann S., Craxton M. A., Coleman H., Fleckenstein B. Primary structure of the herpesvirus saimiri genome. J Virol. 1992 Aug;66(8):5047–5058. doi: 10.1128/jvi.66.8.5047-5058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Block T., Jordan R. Herpes simplex virus type 1 alpha gene containing plasmids can inhibit expression regulated from an alpha promoter in CV-1 but not HeLa cells. Virus Res. 1988 Nov;11(4):269–279. doi: 10.1016/0168-1702(88)90001-9. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Davison A. J., Taylor P. Genetic relations between varicella-zoster virus and Epstein-Barr virus. J Gen Virol. 1987 Apr;68(Pt 4):1067–1079. doi: 10.1099/0022-1317-68-4-1067. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  10. Flint J., Shenk T. Adenovirus E1A protein paradigm viral transactivator. Annu Rev Genet. 1989;23:141–161. doi: 10.1146/annurev.ge.23.120189.001041. [DOI] [PubMed] [Google Scholar]
  11. Frink R. J., Eisenberg R., Cohen G., Wagner E. K. Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C. J Virol. 1983 Feb;45(2):634–647. doi: 10.1128/jvi.45.2.634-647.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gao M., Knipe D. M. Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants. J Virol. 1993 Feb;67(2):876–885. doi: 10.1128/jvi.67.2.876-885.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Godowski P. J., Knipe D. M. Mutations in the major DNA-binding protein gene of herpes simplex virus type 1 result in increased levels of viral gene expression. J Virol. 1983 Sep;47(3):478–486. doi: 10.1128/jvi.47.3.478-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Goldin A. L., Sandri-Goldin R. M., Levine M., Glorioso J. C. Cloning of herpes simplex virus type 1 sequences representing the whole genome. J Virol. 1981 Apr;38(1):50–58. doi: 10.1128/jvi.38.1.50-58.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  19. Hardwicke M. A., Vaughan P. J., Sekulovich R. E., O'Conner R., Sandri-Goldin R. M. The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule. J Virol. 1989 Nov;63(11):4590–4602. doi: 10.1128/jvi.63.11.4590-4602.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hughes R. G., Jr, Munyon W. H. Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation. J Virol. 1975 Aug;16(2):275–283. doi: 10.1128/jvi.16.2.275-283.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  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. McNabb D. S., Courtney R. J. Characterization of the large tegument protein (ICP1/2) of herpes simplex virus type 1. Virology. 1992 Sep;190(1):221–232. doi: 10.1016/0042-6822(92)91208-c. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Nicholas J., Gompels U. A., Craxton M. A., Honess R. W. Conservation of sequence and function between the product of the 52-kilodalton immediate-early gene of herpesvirus saimiri and the BMLF1-encoded transcriptional effector (EB2) of Epstein-Barr virus. J Virol. 1988 Sep;62(9):3250–3257. doi: 10.1128/jvi.62.9.3250-3257.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. Rice S. A., Lam V., Knipe D. M. The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function. J Virol. 1993 Apr;67(4):1778–1787. doi: 10.1128/jvi.67.4.1778-1787.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Sekulovich R. E., Leary K., Sandri-Goldin R. M. The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. J Virol. 1988 Dec;62(12):4510–4522. doi: 10.1128/jvi.62.12.4510-4522.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Smith I. L., Hardwicke M. A., Sandri-Goldin R. M. Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression. Virology. 1992 Jan;186(1):74–86. doi: 10.1016/0042-6822(92)90062-t. [DOI] [PubMed] [Google Scholar]
  43. Smith I. L., Sekulovich R. E., Hardwicke M. A., Sandri-Goldin R. M. Mutations in the activation region of herpes simplex virus regulatory protein ICP27 can be trans dominant. J Virol. 1991 Jul;65(7):3656–3666. doi: 10.1128/jvi.65.7.3656-3666.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Weinheimer S. P., Boyd B. A., Durham S. K., Resnick J. L., O'Boyle D. R., 2nd Deletion of the VP16 open reading frame of herpes simplex virus type 1. J Virol. 1992 Jan;66(1):258–269. doi: 10.1128/jvi.66.1.258-269.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Zhao Y., Holden V. R., Harty R. N., O'Callaghan D. J. Identification and transcriptional analyses of the UL3 and UL4 genes of equine herpesvirus 1, homologs of the ICP27 and glycoprotein K genes of herpes simplex virus. J Virol. 1992 Sep;66(9):5363–5372. doi: 10.1128/jvi.66.9.5363-5372.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES