Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Oct;71(10):7791–7798. doi: 10.1128/jvi.71.10.7791-7798.1997

Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity.

Y T Hwang 1, B Y Liu 1, D M Coen 1, C B Hwang 1
PMCID: PMC192131  PMID: 9311864

Abstract

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3'-5' exonuclease activities, contains sequence motifs that are homologous to those important for 3'-5' exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3'-5' exonuclease contributes substantially to the fidelity of viral DNA replication.

Full Text

The Full Text of this article is available as a PDF (446.3 KB).

Selected References

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

  1. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  2. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  3. Blanco L., Bernad A., Blasco M. A., Salas M. A general structure for DNA-dependent DNA polymerases. Gene. 1991 Apr;100:27–38. doi: 10.1016/0378-1119(91)90346-d. [DOI] [PubMed] [Google Scholar]
  4. Blanco L., Bernad A., Salas M. Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases. Gene. 1992 Mar 1;112(1):139–144. doi: 10.1016/0378-1119(92)90316-h. [DOI] [PubMed] [Google Scholar]
  5. Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chung D. W., Zhang J. A., Tan C. K., Davie E. W., So A. G., Downey K. M. Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11197–11201. doi: 10.1073/pnas.88.24.11197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Davison A. J., Scott J. E. The complete DNA sequence of varicella-zoster virus. J Gen Virol. 1986 Sep;67(Pt 9):1759–1816. doi: 10.1099/0022-1317-67-9-1759. [DOI] [PubMed] [Google Scholar]
  11. Derbyshire V., Freemont P. S., Sanderson M. R., Beese L., Friedman J. M., Joyce C. M., Steitz T. A. Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I. Science. 1988 Apr 8;240(4849):199–201. doi: 10.1126/science.2832946. [DOI] [PubMed] [Google Scholar]
  12. Derbyshire V., Grindley N. D., Joyce C. M. The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 1991 Jan;10(1):17–24. doi: 10.1002/j.1460-2075.1991.tb07916.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Derse D., Bastow K. F., Cheng Y. Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid. J Biol Chem. 1982 Sep 10;257(17):10251–10260. [PubMed] [Google Scholar]
  14. Digard P., Bebrin W. R., Weisshart K., Coen D. M. The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication. J Virol. 1993 Jan;67(1):398–406. doi: 10.1128/jvi.67.1.398-406.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Earl P. L., Jones E. V., Moss B. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659–3663. doi: 10.1073/pnas.83.11.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Echols H., Goodman M. F. Fidelity mechanisms in DNA replication. Annu Rev Biochem. 1991;60:477–511. doi: 10.1146/annurev.bi.60.070191.002401. [DOI] [PubMed] [Google Scholar]
  17. Foury F., Vanderstraeten S. Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. EMBO J. 1992 Jul;11(7):2717–2726. doi: 10.1002/j.1460-2075.1992.tb05337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Freemont P. S., Friedman J. M., Beese L. S., Sanderson M. R., Steitz T. A. Cocrystal structure of an editing complex of Klenow fragment with DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8924–8928. doi: 10.1073/pnas.85.23.8924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Frey M. W., Nossal N. G., Capson T. L., Benkovic S. J. Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2579–2583. doi: 10.1073/pnas.90.7.2579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gibbs J. S., Chiou H. C., Bastow K. F., Cheng Y. C., Coen D. M. Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6672–6676. doi: 10.1073/pnas.85.18.6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gibbs J. S., Chiou H. C., Hall J. D., Mount D. W., Retondo M. J., Weller S. K., Coen D. M. Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7969–7973. doi: 10.1073/pnas.82.23.7969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  24. 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]
  25. Hall J. D., Almy R. E. Evidence for control of herpes simplex virus mutagenesis by the viral DNA polymerase. Virology. 1982 Jan 30;116(2):535–543. doi: 10.1016/0042-6822(82)90146-5. [DOI] [PubMed] [Google Scholar]
  26. Hall J. D., Coen D. M., Fisher B. L., Weisslitz M., Randall S., Almy R. E., Gelep P. T., Schaffer P. A. Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus. Virology. 1984 Jan 15;132(1):26–37. doi: 10.1016/0042-6822(84)90088-6. [DOI] [PubMed] [Google Scholar]
  27. Hall J. D., Orth K. L., Sander K. L., Swihart B. M., Senese R. A. Mutations within conserved motifs in the 3'-5' exonuclease domain of herpes simplex virus DNA polymerase. J Gen Virol. 1995 Dec;76(Pt 12):2999–3008. doi: 10.1099/0022-1317-76-12-2999. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Hwang C. B., Chen H. J. An altered spectrum of herpes simplex virus mutations mediated by an antimutator DNA polymerase. Gene. 1995 Jan 23;152(2):191–193. doi: 10.1016/0378-1119(94)00712-2. [DOI] [PubMed] [Google Scholar]
  30. Hwang C. B., Horsburgh B., Pelosi E., Roberts S., Digard P., Coen D. M. A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5461–5465. doi: 10.1073/pnas.91.12.5461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hwang C. B., Ruffner K. L., Coen D. M. A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance. J Virol. 1992 Mar;66(3):1774–1776. doi: 10.1128/jvi.66.3.1774-1776.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hwang C. B., Shillitoe E. J. DNA sequence of mutations induced in cells by herpes simplex virus type-1. Virology. 1990 Sep;178(1):180–188. doi: 10.1016/0042-6822(90)90392-5. [DOI] [PubMed] [Google Scholar]
  33. Joyce C. M., Kelley W. S., Grindley N. D. Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I. J Biol Chem. 1982 Feb 25;257(4):1958–1964. [PubMed] [Google Scholar]
  34. Knopf C. W., Weisshart K. Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase. Eur J Biochem. 1990 Jul 31;191(2):263–273. doi: 10.1111/j.1432-1033.1990.tb19119.x. [DOI] [PubMed] [Google Scholar]
  35. Knopf K. W. Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity. Eur J Biochem. 1979 Jul;98(1):231–244. doi: 10.1111/j.1432-1033.1979.tb13181.x. [DOI] [PubMed] [Google Scholar]
  36. Kunkel T. A. Exonucleolytic proofreading. Cell. 1988 Jun 17;53(6):837–840. doi: 10.1016/s0092-8674(88)90189-4. [DOI] [PubMed] [Google Scholar]
  37. Kühn F. J., Knopf C. W. Herpes simplex virus type 1 DNA polymerase. Mutational analysis of the 3'-5'-exonuclease domain. J Biol Chem. 1996 Nov 15;271(46):29245–29254. doi: 10.1074/jbc.271.46.29245. [DOI] [PubMed] [Google Scholar]
  38. Larder B. A., Kemp S. D., Darby G. Related functional domains in virus DNA polymerases. EMBO J. 1987 Jan;6(1):169–175. doi: 10.1002/j.1460-2075.1987.tb04735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Morrison A., Bell J. B., Kunkel T. A., Sugino A. Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9473–9477. doi: 10.1073/pnas.88.21.9473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Reha-Krantz L. J., Nonay R. L. Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity. J Biol Chem. 1993 Dec 25;268(36):27100–27108. [PubMed] [Google Scholar]
  45. Reha-Krantz L. J., Stocki S., Nonay R. L., Dimayuga E., Goodrich L. D., Konigsberg W. H., Spicer E. K. DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2417–2421. doi: 10.1073/pnas.88.6.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Simon M., Giot L., Faye G. The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J. 1991 Aug;10(8):2165–2170. doi: 10.1002/j.1460-2075.1991.tb07751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Soengas M. S., Esteban J. A., Lázaro J. M., Bernad A., Blasco M. A., Salas M., Blanco L. Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities. EMBO J. 1992 Nov;11(11):4227–4237. doi: 10.1002/j.1460-2075.1992.tb05517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Spicer E. K., Rush J., Fung C., Reha-Krantz L. J., Karam J. D., Konigsberg W. H. Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases. J Biol Chem. 1988 Jun 5;263(16):7478–7486. [PubMed] [Google Scholar]
  49. Vialard J., Lalumière M., Vernet T., Briedis D., Alkhatib G., Henning D., Levin D., Richardson C. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene. J Virol. 1990 Jan;64(1):37–50. doi: 10.1128/jvi.64.1.37-50.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang T. S., Wong S. W., Korn D. Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 1989 Jan;3(1):14–21. doi: 10.1096/fasebj.3.1.2642867. [DOI] [PubMed] [Google Scholar]
  51. Weisshart K., Kuo A. A., Hwang C. B., Kumura K., Coen D. M. Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis. J Biol Chem. 1994 Sep 9;269(36):22788–22796. [PubMed] [Google Scholar]
  52. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshikawa H., Ito J. Nucleotide sequence of the major early region of bacteriophage phi 29. Gene. 1982 Mar;17(3):323–335. doi: 10.1016/0378-1119(82)90149-4. [DOI] [PubMed] [Google Scholar]
  54. Zhang J., Chung D. W., Tan C. K., Downey K. M., Davie E. W., So A. G. Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases. Biochemistry. 1991 Dec 24;30(51):11742–11750. doi: 10.1021/bi00115a002. [DOI] [PubMed] [Google Scholar]

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

RESOURCES