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. 1997 Apr;71(4):3146–3160. doi: 10.1128/jvi.71.4.3146-3160.1997

Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays.

L Zhong 1, G S Hayward 1
PMCID: PMC191447  PMID: 9060678

Abstract

Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.

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Selected References

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  1. Applegren N., Hickey R. J., Kleinschmidt A. M., Zhou Q., Coll J., Wills P., Swaby R., Wei Y., Quan J. Y., Lee M. Y. Further characterization of the human cell multiprotein DNA replication complex. J Cell Biochem. 1995 Sep;59(1):91–107. doi: 10.1002/jcb.240590111. [DOI] [PubMed] [Google Scholar]
  2. Bargonetti J., Friedman P. N., Kern S. E., Vogelstein B., Prives C. Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell. 1991 Jun 14;65(6):1083–1091. doi: 10.1016/0092-8674(91)90560-l. [DOI] [PubMed] [Google Scholar]
  3. Boehmer P. E., Craigie M. C., Stow N. D., Lehman I. R. Association of origin binding protein and single strand DNA-binding protein, ICP8, during herpes simplex virus type 1 DNA replication in vivo. J Biol Chem. 1994 Nov 18;269(46):29329–29334. [PubMed] [Google Scholar]
  4. Boehmer P. E., Lehman I. R. Physical interaction between the herpes simplex virus 1 origin-binding protein and single-stranded DNA-binding protein ICP8. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8444–8448. doi: 10.1073/pnas.90.18.8444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brénot-Bosc F., Gupta S., Margolis R. L., Fotedar R. Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. Chromosoma. 1995 Feb;103(8):517–527. doi: 10.1007/BF00355316. [DOI] [PubMed] [Google Scholar]
  6. Bush M., Yager D. R., Gao M., Weisshart K., Marcy A. I., Coen D. M., Knipe D. M. Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein. J Virol. 1991 Mar;65(3):1082–1089. doi: 10.1128/jvi.65.3.1082-1089.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calder J. M., Stow E. C., Stow N. D. On the cellular localization of the components of the herpes simplex virus type 1 helicase-primase complex and the viral origin-binding protein. J Gen Virol. 1992 Mar;73(Pt 3):531–538. doi: 10.1099/0022-1317-73-3-531. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chan Y. J., Chiou C. J., Huang Q., Hayward G. S. Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. J Virol. 1996 Dec;70(12):8590–8605. doi: 10.1128/jvi.70.12.8590-8605.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chiou H. C., Weller S. K., Coen D. M. Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors. Virology. 1985 Sep;145(2):213–226. doi: 10.1016/0042-6822(85)90155-2. [DOI] [PubMed] [Google Scholar]
  11. Cox L. S., Hupp T., Midgley C. A., Lane D. P. A direct effect of activated human p53 on nuclear DNA replication. EMBO J. 1995 May 1;14(9):2099–2105. doi: 10.1002/j.1460-2075.1995.tb07201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crute J. J., Tsurumi T., Zhu L. A., Weller S. K., Olivo P. D., Challberg M. D., Mocarski E. S., Lehman I. R. Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2186–2189. doi: 10.1073/pnas.86.7.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Curtin K. D., Knipe D. M. Altered properties of the herpes simplex virus ICP8 DNA-binding protein in cells infected with ICP27 mutant viruses. Virology. 1993 Sep;196(1):1–14. doi: 10.1006/viro.1993.1449. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Dutta A., Ruppert J. M., Aster J. C., Winchester E. Inhibition of DNA replication factor RPA by p53. Nature. 1993 Sep 2;365(6441):79–82. doi: 10.1038/365079a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Goodrich L. D., Schaffer P. A., Dorsky D. I., Crumpacker C. S., Parris D. S. Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis. J Virol. 1990 Dec;64(12):5738–5749. doi: 10.1128/jvi.64.12.5738-5749.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartwell L. H., Kastan M. B. Cell cycle control and cancer. Science. 1994 Dec 16;266(5192):1821–1828. doi: 10.1126/science.7997877. [DOI] [PubMed] [Google Scholar]
  21. Heilbronn R., zur Hausen H. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome. J Virol. 1989 Sep;63(9):3683–3692. doi: 10.1128/jvi.63.9.3683-3692.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hozák P., Hassan A. B., Jackson D. A., Cook P. R. Visualization of replication factories attached to nucleoskeleton. Cell. 1993 Apr 23;73(2):361–373. doi: 10.1016/0092-8674(93)90235-i. [DOI] [PubMed] [Google Scholar]
  23. Jackson D. A. S-phase progression in synchronized human cells. Exp Cell Res. 1995 Sep;220(1):62–70. doi: 10.1006/excr.1995.1292. [DOI] [PubMed] [Google Scholar]
  24. Kanda T., Segawa K., Ohuchi N., Mori S., Ito Y. Stimulation of polyomavirus DNA replication by wild-type p53 through the DNA-binding site. Mol Cell Biol. 1994 Apr;14(4):2651–2663. doi: 10.1128/mcb.14.4.2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  26. Knipe D. M., Senechek D., Rice S. A., Smith J. L. Stages in the nuclear association of the herpes simplex virus transcriptional activator protein ICP4. J Virol. 1987 Feb;61(2):276–284. doi: 10.1128/jvi.61.2.276-284.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knipe D. M., Smith J. L. A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins. Mol Cell Biol. 1986 Jul;6(7):2371–2381. doi: 10.1128/mcb.6.7.2371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knipe D. M. The role of viral and cellular nuclear proteins in herpes simplex virus replication. Adv Virus Res. 1989;37:85–123. doi: 10.1016/s0065-3527(08)60833-7. [DOI] [PubMed] [Google Scholar]
  29. Liptak L. M., Uprichard S. L., Knipe D. M. Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures. J Virol. 1996 Mar;70(3):1759–1767. doi: 10.1128/jvi.70.3.1759-1767.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lukonis C. J., Weller S. K. Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication. J Virol. 1996 Mar;70(3):1751–1758. doi: 10.1128/jvi.70.3.1751-1758.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lukonis C. J., Weller S. K. The herpes simplex virus type 1 transactivator ICPO mediates aberrant intracellular localization of the viral helicase/primase complex subunits. Virology. 1996 Jun 15;220(2):495–501. doi: 10.1006/viro.1996.0338. [DOI] [PubMed] [Google Scholar]
  32. Maul G. G., Ishov A. M., Everett R. D. Nuclear domain 10 as preexisting potential replication start sites of herpes simplex virus type-1. Virology. 1996 Mar 1;217(1):67–75. doi: 10.1006/viro.1996.0094. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Miller S. D., Farmer G., Prives C. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner. Mol Cell Biol. 1995 Dec;15(12):6554–6560. doi: 10.1128/mcb.15.12.6554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mullen M. A., Gerstberger S., Ciufo D. M., Mosca J. D., Hayward G. S. Evaluation of colocalization interactions between the IE110, IE175, and IE63 transactivator proteins of herpes simplex virus within subcellular punctate structures. J Virol. 1995 Jan;69(1):476–491. doi: 10.1128/jvi.69.1.476-491.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Olivo P. D., Nelson N. J., Challberg M. D. Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression. J Virol. 1989 Jan;63(1):196–204. doi: 10.1128/jvi.63.1.196-204.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Persson R. H., Bacchetti S., Smiley J. R. Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans. J Virol. 1985 May;54(2):414–421. doi: 10.1128/jvi.54.2.414-421.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Phelan A., Carmo-Fonseca M., McLaughlan J., Lamond A. I., Clements J. B. A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9056–9060. doi: 10.1073/pnas.90.19.9056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pizzorno M. C., Hayward G. S. The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol. 1990 Dec;64(12):6154–6165. doi: 10.1128/jvi.64.12.6154-6165.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Randall R. E., Dinwoodie N. Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP 4 is associated with progeny virus DNA. J Gen Virol. 1986 Oct;67(Pt 10):2163–2177. doi: 10.1099/0022-1317-67-10-2163. [DOI] [PubMed] [Google Scholar]
  45. Rixon F. J., Atkinson M. A., Hay J. Intranuclear distribution of herpes simplex virus type 2 DNA synthesis: examination by light and electron microscopy. J Gen Virol. 1983 Sep;64(Pt 9):2087–2092. doi: 10.1099/0022-1317-64-9-2087. [DOI] [PubMed] [Google Scholar]
  46. Ruyechan W. T., Weir A. C. Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. J Virol. 1984 Dec;52(3):727–733. doi: 10.1128/jvi.52.3.727-733.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sandri-Goldin R. M., Hibbard M. K., Hardwicke M. A. The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing. J Virol. 1995 Oct;69(10):6063–6076. doi: 10.1128/jvi.69.10.6063-6076.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sandri-Goldin R. M., Hibbard M. K. The herpes simplex virus type 1 regulatory protein ICP27 coimmunoprecipitates with anti-Sm antiserum, and the C terminus appears to be required for this interaction. J Virol. 1996 Jan;70(1):108–118. doi: 10.1128/jvi.70.1.108-118.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sarisky R. T., Hayward G. S. Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays. J Virol. 1996 Nov;70(11):7398–7413. doi: 10.1128/jvi.70.11.7398-7413.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Uprichard S. L., Knipe D. M. Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes. J Virol. 1996 Mar;70(3):1969–1980. doi: 10.1128/jvi.70.3.1969-1980.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Weir H. M., Stow N. D. Two binding sites for the herpes simplex virus type 1 UL9 protein are required for efficient activity of the oriS replication origin. J Gen Virol. 1990 Jun;71(Pt 6):1379–1385. doi: 10.1099/0022-1317-71-6-1379. [DOI] [PubMed] [Google Scholar]
  54. Wilcock D., Lane D. P. Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature. 1991 Jan 31;349(6308):429–431. doi: 10.1038/349429a0. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Zhu L. A., Weller S. K. The UL5 gene of herpes simplex virus type 1: isolation of a lacZ insertion mutant and association of the UL5 gene product with other members of the helicase-primase complex. J Virol. 1992 Jan;66(1):458–468. doi: 10.1128/jvi.66.1.458-468.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhu L. A., Weller S. K. The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function. J Virol. 1992 Jan;66(1):469–479. doi: 10.1128/jvi.66.1.469-479.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. de Bruyn Kops A., Knipe D. M. Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures. J Virol. 1994 Jun;68(6):3512–3526. doi: 10.1128/jvi.68.6.3512-3526.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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