Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Jun;68(6):3693–3701. doi: 10.1128/jvi.68.6.3693-3701.1994

Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity.

D K Klinedinst 1, M D Challberg 1
PMCID: PMC236874  PMID: 8189507

Abstract

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.

Full text

PDF
3693

Images in this article

3696Image
on p.3696
3697Image
on p.3697
3697Image
on p.3697
3698Image
on p.3698
3698Image
on p.3698
3699Image
on p.3699

Selected References

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

  1. Arai K., Kornberg A. A general priming system employing only dnaB protein and primase for DNA replication. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4308–4312. doi: 10.1073/pnas.76.9.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Argos P. A sequence motif in many polymerases. Nucleic Acids Res. 1988 Nov 11;16(21):9909–9916. doi: 10.1093/nar/16.21.9909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernad A., Blanco L., Salas M. Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase. Gene. 1990 Sep 28;94(1):45–51. doi: 10.1016/0378-1119(90)90466-5. [DOI] [PubMed] [Google Scholar]
  4. Bernstein J. A., Richardson C. C. A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity. Proc Natl Acad Sci U S A. 1988 Jan;85(2):396–400. doi: 10.1073/pnas.85.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Carmichael E. P., Kosovsky M. J., Weller S. K. Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis. J Virol. 1988 Jan;62(1):91–99. doi: 10.1128/jvi.62.1.91-99.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carmichael E. P., Weller S. K. Herpes simplex virus type 1 DNA synthesis requires the product of the UL8 gene: isolation and characterization of an ICP6::lacZ insertion mutation. J Virol. 1989 Feb;63(2):591–599. doi: 10.1128/jvi.63.2.591-599.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chartrand P., Crumpacker C. S., Schaffer P. A., Wilkie N. M. Physical and genetic analysis of the herpes simplex virus DNA polymerase locus. Virology. 1980 Jun;103(2):311–326. doi: 10.1016/0042-6822(80)90190-7. [DOI] [PubMed] [Google Scholar]
  11. Coen D. M., Aschman D. P., Gelep P. T., Retondo M. J., Weller S. K., Schaffer P. A. Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus. J Virol. 1984 Jan;49(1):236–247. doi: 10.1128/jvi.49.1.236-247.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crute J. J., Lehman I. R. Herpes simplex virus-1 helicase-primase. Physical and catalytic properties. J Biol Chem. 1991 Mar 5;266(7):4484–4488. [PubMed] [Google Scholar]
  13. 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]
  14. Crute J. J., Mocarski E. S., Lehman I. R. A DNA helicase induced by herpes simplex virus type 1. Nucleic Acids Res. 1988 Jul 25;16(14A):6585–6596. doi: 10.1093/nar/16.14.6585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Date T., Yamamoto S., Tanihara K., Nishimoto Y., Matsukage A. Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding. Biochemistry. 1991 May 28;30(21):5286–5292. doi: 10.1021/bi00235a023. [DOI] [PubMed] [Google Scholar]
  17. Dodson M. S., Crute J. J., Bruckner R. C., Lehman I. R. Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells. J Biol Chem. 1989 Dec 15;264(35):20835–20838. [PubMed] [Google Scholar]
  18. 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]
  19. Elias P., Lehman I. R. Interaction of origin binding protein with an origin of replication of herpes simplex virus 1. Proc Natl Acad Sci U S A. 1988 May;85(9):2959–2963. doi: 10.1073/pnas.85.9.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Goldstein D. J., Weller S. K. An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J Virol. 1988 Aug;62(8):2970–2977. doi: 10.1128/jvi.62.8.2970-2977.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Purification and properties of the gene 4 protein of bacteriophage T7. J Biol Chem. 1975 Jul 25;250(14):5523–5529. [PubMed] [Google Scholar]
  29. Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
  30. Ilyina T. V., Gorbalenya A. E., Koonin E. V. Organization and evolution of bacterial and bacteriophage primase-helicase systems. J Mol Evol. 1992 Apr;34(4):351–357. doi: 10.1007/BF00160243. [DOI] [PubMed] [Google Scholar]
  31. Jablonski S. A., Luo M., Morrow C. D. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol. 1991 Sep;65(9):4565–4572. doi: 10.1128/jvi.65.9.4565-4572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Labeit S., Lehrach H., Goody R. S. DNA sequencing using alpha-thiodeoxynucleotides. Methods Enzymol. 1987;155:166–177. doi: 10.1016/0076-6879(87)55015-7. [DOI] [PubMed] [Google Scholar]
  33. LeBowitz J. H., McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986 Apr 5;261(10):4738–4748. [PubMed] [Google Scholar]
  34. 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]
  35. Marcy A. I., Hwang C. B., Ruffner K. L., Coen D. M. Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition. J Virol. 1990 Dec;64(12):5883–5890. doi: 10.1128/jvi.64.12.5883-5890.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Martinez R., Shao L., Weller S. K. The conserved helicase motifs of the herpes simplex virus type 1 origin-binding protein UL9 are important for function. J Virol. 1992 Nov;66(11):6735–6746. doi: 10.1128/jvi.66.11.6735-6746.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McGeoch D. J., Dalrymple M. A., Dolan A., McNab D., Perry L. J., Taylor P., Challberg M. D. Structures of herpes simplex virus type 1 genes required for replication of virus DNA. J Virol. 1988 Feb;62(2):444–453. doi: 10.1128/jvi.62.2.444-453.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mendelman L. V., Richardson C. C. Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein. J Biol Chem. 1991 Dec 5;266(34):23240–23250. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. 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]
  44. Purifoy D. J., Powell K. L. Temperature-sensitive mutants in two distinct complementation groups of herpes simplex virus type 1 specify thermolabile DNA polymerase. J Gen Virol. 1981 May;54(Pt 1):219–222. doi: 10.1099/0022-1317-54-1-219. [DOI] [PubMed] [Google Scholar]
  45. Scherzinger E., Lanka E., Morelli G., Seiffert D., Yuki A. Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. Eur J Biochem. 1977 Feb;72(3):543–558. doi: 10.1111/j.1432-1033.1977.tb11278.x. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Strack B., Lessl M., Calendar R., Lanka E. A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. J Biol Chem. 1992 Jun 25;267(18):13062–13072. [PubMed] [Google Scholar]
  49. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  50. Venkatesan M., Silver L. L., Nossal N. G. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase. J Biol Chem. 1982 Oct 25;257(20):12426–12434. [PubMed] [Google Scholar]
  51. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weller S. K., Carmichael E. P., Aschman D. P., Goldstein D. J., Schaffer P. A. Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA. Virology. 1987 Nov;161(1):198–210. doi: 10.1016/0042-6822(87)90186-3. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. 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]

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

RESOURCES