Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1993 Oct;67(10):5922–5931. doi: 10.1128/jvi.67.10.5922-5931.1993

Two regions of the herpes simplex virus type 1 UL42 protein are required for its functional interaction with the viral DNA polymerase.

S J Monahan 1, T F Barlam 1, C S Crumpacker 1, D S Parris 1
PMCID: PMC238012  PMID: 8396660

Abstract

Two essential gene products of herpes simplex virus type 1, the viral DNA polymerase (pol) and UL42, its accessory protein, physically and functionally interact to form the core of the viral DNA replication complex. Understanding this essential interaction would provide a basis from which to develop novel anti-herpesvirus agents. We previously have shown that when coexpressed in an in vitro transcription-translation system, UL42 stimulates pol activity (M. L. Gallo, D. I. Dorsky, C. S. Crumpacker, and D. S. Parris, J. Virol. 63:5023-5029, 1989). By analyzing various insertion, deletion, and frameshift mutations of UL42 in this system, we found the C-terminal 149 amino acids to be dispensable for the ability of the protein to stimulate pol activity. In addition, two distinct internal regions of UL42 were found to be required for pol stimulation. Regions I and II were defined to lie between amino acid residues 129 and 163 and between residues 202 and 337, respectively. When physical association was examined with antibody to UL42, pol was found to coimmunoprecipitate to the same level when expressed with a UL42 mutant protein lacking region I as that with wild-type UL42. Thus, mere physical association is insufficient for stimulation of pol activity. Deletion of region II reduced or eliminated coimmunoprecipitation with pol. Interestingly, an antibody to pol specific for residues 1216 to 1224 coimmunoprecipitated UL42 when both proteins were synthesized in a baculovirus expression system but not in rabbit reticulocyte lysates. These results indicate that (i) at least a portion of the region recognized by the pol antiserum may be accessible in the pol-UL42 heterodimer and (ii) immunoprecipitation results for products made in different expression systems may vary. Thus, at least two distinct regions of UL42 are essential for functional interaction with pol. Moreover, these results point to a UL42 region I function other than physical association with pol.

Full text

PDF
5922

Images in this article

5926Image
on p.5926
5927Image
on p.5927
5928Image
on p.5928

Selected References

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

  1. Bruckner R. C., Crute J. J., Dodson M. S., Lehman I. R. The herpes simplex virus 1 origin binding protein: a DNA helicase. J Biol Chem. 1991 Feb 5;266(4):2669–2674. [PubMed] [Google Scholar]
  2. 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]
  3. Calder J. M., Stow N. D. Herpes simplex virus helicase-primase: the UL8 protein is not required for DNA-dependent ATPase and DNA helicase activities. Nucleic Acids Res. 1990 Jun 25;18(12):3573–3578. doi: 10.1093/nar/18.12.3573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  5. Chartrand P., Stow N. D., Timbury M. C., Wilkie N. M. Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue. J Virol. 1979 Aug;31(2):265–276. doi: 10.1128/jvi.31.2.265-276.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Crute J. J., Lehman I. R. Herpes simplex-1 DNA polymerase. Identification of an intrinsic 5'----3' exonuclease with ribonuclease H activity. J Biol Chem. 1989 Nov 15;264(32):19266–19270. [PubMed] [Google Scholar]
  9. 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]
  10. Digard P., Chow C. S., Pirrit L., Coen D. M. Functional analysis of the herpes simplex virus UL42 protein. J Virol. 1993 Mar;67(3):1159–1168. doi: 10.1128/jvi.67.3.1159-1168.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Digard P., Coen D. M. A novel functional domain of an alpha-like DNA polymerase. The binding site on the herpes simplex virus polymerase for the viral UL42 protein. J Biol Chem. 1990 Oct 15;265(29):17393–17396. [PubMed] [Google Scholar]
  12. Dodson M. S., Lehman I. R. Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1105–1109. doi: 10.1073/pnas.88.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dorsky D. I., Crumpacker C. S. Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity. J Virol. 1988 Sep;62(9):3224–3232. doi: 10.1128/jvi.62.9.3224-3232.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elias P., O'Donnell M. E., Mocarski E. S., Lehman I. R. A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6322–6326. doi: 10.1073/pnas.83.17.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fierer D. S., Challberg M. D. Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein. J Virol. 1992 Jul;66(7):3986–3995. doi: 10.1128/jvi.66.7.3986-3995.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gallo M. L., Dorsky D. I., Crumpacker C. S., Parris D. S. The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase. J Virol. 1989 Dec;63(12):5023–5029. doi: 10.1128/jvi.63.12.5023-5029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gallo M. L., Jackwood D. H., Murphy M., Marsden H. S., Parris D. S. Purification of the herpes simplex virus type 1 65-kilodalton DNA-binding protein: properties of the protein and evidence of its association with the virus-encoded DNA polymerase. J Virol. 1988 Aug;62(8):2874–2883. doi: 10.1128/jvi.62.8.2874-2883.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  19. Gibbs J. S., Weisshart K., Digard P., deBruynKops A., Knipe D. M., Coen D. M. Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site. Mol Cell Biol. 1991 Sep;11(9):4786–4795. doi: 10.1128/mcb.11.9.4786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goodrich L. D., Rixon F. J., Parris D. S. Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1. J Virol. 1989 Jan;63(1):137–147. doi: 10.1128/jvi.63.1.137-147.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gottlieb J., Marcy A. I., Coen D. M., Challberg M. D. The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity. J Virol. 1990 Dec;64(12):5976–5987. doi: 10.1128/jvi.64.12.5976-5987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haffey M. L., Stevens J. T., Terry B. J., Dorsky D. I., Crumpacker C. S., Wietstock S. M., Ruyechan W. T., Field A. K. Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. J Virol. 1988 Dec;62(12):4493–4498. doi: 10.1128/jvi.62.12.4493-4498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hernandez T. R., Lehman I. R. Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein. J Biol Chem. 1990 Jul 5;265(19):11227–11232. [PubMed] [Google Scholar]
  25. Jofre J. T., Schaffer P. A., Parris D. S. Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1. J Virol. 1977 Sep;23(3):833–836. doi: 10.1128/jvi.23.3.833-836.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson P. A., Best M. G., Friedmann T., Parris D. S. Isolation of a herpes simplex virus type 1 mutant deleted for the essential UL42 gene and characterization of its null phenotype. J Virol. 1991 Feb;65(2):700–710. doi: 10.1128/jvi.65.2.700-710.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marchetti M. E., Smith C. A., Schaffer P. A. A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL. J Virol. 1988 Mar;62(3):715–721. doi: 10.1128/jvi.62.3.715-721.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marcy A. I., Olivo P. D., Challberg M. D., Coen D. M. Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells. Nucleic Acids Res. 1990 Mar 11;18(5):1207–1215. doi: 10.1093/nar/18.5.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marcy A. I., Yager D. R., Coen D. M. Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus. J Virol. 1990 May;64(5):2208–2216. doi: 10.1128/jvi.64.5.2208-2216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marians K. J. Prokaryotic DNA replication. Annu Rev Biochem. 1992;61:673–719. doi: 10.1146/annurev.bi.61.070192.003325. [DOI] [PubMed] [Google Scholar]
  31. Marsden H. S., Campbell M. E., Haarr L., Frame M. C., Parris D. S., Murphy M., Hope R. G., Muller M. T., Preston C. M. The 65,000-Mr DNA-binding and virion trans-inducing proteins of herpes simplex virus type 1. J Virol. 1987 Aug;61(8):2428–2437. doi: 10.1128/jvi.61.8.2428-2437.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Murphy M., Schenk P., Lankinen H. M., Cross A. M., Taylor P., Owsianka A., Hope R. G., Ludwig H., Marsden H. S. Mapping of epitopes on the 65k DNA-binding protein of herpes simplex virus type 1. J Gen Virol. 1989 Sep;70(Pt 9):2357–2364. doi: 10.1099/0022-1317-70-9-2357. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Olivo P. D., Nelson N. J., Challberg M. D. Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5414–5418. doi: 10.1073/pnas.85.15.5414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Owsianka A. M., Hart G., Murphy M., Gottlieb J., Boehme R., Challberg M., Marsden H. S. Inhibition of herpes simplex virus type 1 DNA polymerase activity by peptides from the UL42 accessory protein is largely nonspecific. J Virol. 1993 Jan;67(1):258–264. doi: 10.1128/jvi.67.1.258-264.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Pakula A. A., Sauer R. T. Genetic analysis of protein stability and function. Annu Rev Genet. 1989;23:289–310. doi: 10.1146/annurev.ge.23.120189.001445. [DOI] [PubMed] [Google Scholar]
  39. Parris D. S., Cross A., Haarr L., Orr A., Frame M. C., Murphy M., McGeoch D. J., Marsden H. S. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1. J Virol. 1988 Mar;62(3):818–825. doi: 10.1128/jvi.62.3.818-825.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Purifoy D. J., Lewis R. B., Powell K. L. Identification of the herpes simplex virus DNA polymerase gene. Nature. 1977 Oct 13;269(5629):621–623. doi: 10.1038/269621a0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Sherman G., Gottlieb J., Challberg M. D. The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization. J Virol. 1992 Aug;66(8):4884–4892. doi: 10.1128/jvi.66.8.4884-4892.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tenney D. J., Micheletti P. A., Stevens J. T., Hamatake R. K., Matthews J. T., Sanchez A. R., Hurlburt W. W., Bifano M., Cordingley M. G. Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity. J Virol. 1993 Jan;67(1):543–547. doi: 10.1128/jvi.67.1.543-547.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vaughan P. J., Banks L. M., Purifoy D. J., Powell K. L. Interactions between herpes simplex virus DNA-binding proteins. J Gen Virol. 1984 Nov;65(Pt 11):2033–2041. doi: 10.1099/0022-1317-65-11-2033. [DOI] [PubMed] [Google Scholar]
  46. Vaughan P. J., Purifoy D. J., Powell K. L. DNA-binding protein associated with herpes simplex virus DNA polymerase. J Virol. 1985 Feb;53(2):501–508. doi: 10.1128/jvi.53.2.501-508.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Yager D. R., Marcy A. I., Coen D. M. Translational regulation of herpes simplex virus DNA polymerase. J Virol. 1990 May;64(5):2217–2225. doi: 10.1128/jvi.64.5.2217-2225.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. 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]

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

RESOURCES