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. 1996 May;70(5):3169–3175. doi: 10.1128/jvi.70.5.3169-3175.1996

Branched structures in the intracellular DNA of herpes simplex virus type 1.

A Severini 1, D G Scraba 1, D L Tyrrell 1
PMCID: PMC190180  PMID: 8627797

Abstract

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.

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Selected References

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  1. Addison C., Rixon F. J., Palfreyman J. W., O'Hara M., Preston V. G. Characterisation of a herpes simplex virus type 1 mutant which has a temperature-sensitive defect in penetration of cells and assembly of capsids. Virology. 1984 Oct 30;138(2):246–259. doi: 10.1016/0042-6822(84)90349-0. [DOI] [PubMed] [Google Scholar]
  2. Addison C., Rixon F. J., Preston V. G. Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids. J Gen Virol. 1990 Oct;71(Pt 10):2377–2384. doi: 10.1099/0022-1317-71-10-2377. [DOI] [PubMed] [Google Scholar]
  3. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  4. Chou J., Roizman B. Characterization of DNA sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome. J Virol. 1989 Mar;63(3):1059–1068. doi: 10.1128/jvi.63.3.1059-1068.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danovich R. M., Frenkel N. Herpes simplex virus induces the replication of foreign DNA. Mol Cell Biol. 1988 Aug;8(8):3272–3281. doi: 10.1128/mcb.8.8.3272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deiss L. P., Chou J., Frenkel N. Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA. J Virol. 1986 Sep;59(3):605–618. doi: 10.1128/jvi.59.3.605-618.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Delius H., Clements J. B. A partial denaturation map of herpes simplex virus type 1 DNA: evidence for inversions of the unique DNA regions. J Gen Virol. 1976 Oct;33(1):125–133. doi: 10.1099/0022-1317-33-1-125. [DOI] [PubMed] [Google Scholar]
  8. Desai P., DeLuca N. A., Glorioso J. C., Person S. Mutations in herpes simplex virus type 1 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA. J Virol. 1993 Mar;67(3):1357–1364. doi: 10.1128/jvi.67.3.1357-1364.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dickie P., McFadden G., Morgan A. R. The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I. J Biol Chem. 1987 Oct 25;262(30):14826–14836. [PubMed] [Google Scholar]
  10. Dunderdale H. J., Benson F. E., Parsons C. A., Sharples G. J., Lloyd R. G., West S. C. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature. 1991 Dec 19;354(6354):506–510. doi: 10.1038/354506a0. [DOI] [PubMed] [Google Scholar]
  11. Dutch R. E., Bianchi V., Lehman I. R. Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences. J Virol. 1995 May;69(5):3084–3089. doi: 10.1128/jvi.69.5.3084-3089.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dutch R. E., Bruckner R. C., Mocarski E. S., Lehman I. R. Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences. J Virol. 1992 Jan;66(1):277–285. doi: 10.1128/jvi.66.1.277-285.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dutch R. E., Zemelman B. V., Lehman I. R. Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination. J Virol. 1994 Jun;68(6):3733–3741. doi: 10.1128/jvi.68.6.3733-3741.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Futcher A. B. Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae. J Theor Biol. 1986 Mar 21;119(2):197–204. doi: 10.1016/s0022-5193(86)80074-1. [DOI] [PubMed] [Google Scholar]
  15. Garber D. A., Beverley S. M., Coen D. M. Demonstration of circularization of herpes simplex virus DNA following infection using pulsed field gel electrophoresis. Virology. 1993 Nov;197(1):459–462. doi: 10.1006/viro.1993.1612. [DOI] [PubMed] [Google Scholar]
  16. Hayward G. S., Jacob R. J., Wadsworth S. C., Roizman B. Anatomy of herpes simplex virus DNA: evidence for four populations of molecules that differ in the relative orientations of their long and short components. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4243–4247. doi: 10.1073/pnas.72.11.4243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffmann P. J., Cheng Y. C. The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme. J Biol Chem. 1978 May 25;253(10):3557–3562. [PubMed] [Google Scholar]
  18. Hoffmann P. J. Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus. J Virol. 1981 Jun;38(3):1005–1014. doi: 10.1128/jvi.38.3.1005-1014.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacob R. J., Morse L. S., Roizman B. Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. J Virol. 1979 Feb;29(2):448–457. doi: 10.1128/jvi.29.2.448-457.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacob R. J., Roizman B. Anatomy of herpes simplex virus DNA VIII. Properties of the replicating DNA. J Virol. 1977 Aug;23(2):394–411. doi: 10.1128/jvi.23.2.394-411.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jongeneel C. V., Bachenheimer S. L. Structure of replicating herpes simplex virus DNA. J Virol. 1981 Aug;39(2):656–660. doi: 10.1128/jvi.39.2.656-660.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lurz R., Grote M., Dijk J., Reinhardt R., Dobrinski B. Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius. EMBO J. 1986 Dec 20;5(13):3715–3721. doi: 10.1002/j.1460-2075.1986.tb04705.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Mocarski E. S., Roizman B. Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5626–5630. doi: 10.1073/pnas.79.18.5626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morgan A. R., Evans D. H., Lee J. S., Pulleyblank D. E. Review: ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions. Nucleic Acids Res. 1979 Oct 10;7(3):571–594. doi: 10.1093/nar/7.3.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morgan A. R., Severini A. Interconversion of replication and recombination structures: implications for terminal repeats and concatemers. J Theor Biol. 1990 May 22;144(2):195–202. doi: 10.1016/s0022-5193(05)80318-2. [DOI] [PubMed] [Google Scholar]
  27. Mosig G. The essential role of recombination in phage T4 growth. Annu Rev Genet. 1987;21:347–371. doi: 10.1146/annurev.ge.21.120187.002023. [DOI] [PubMed] [Google Scholar]
  28. Ooka T., De Turenne M., De The G., Daillie J. Epstein-Barr virus-specific DNase activity in nonproducer Raji cells after treatment with 12-o-tetradecanoylphorbol-13-acetate and sodium butyrate. J Virol. 1984 Feb;49(2):626–628. doi: 10.1128/jvi.49.2.626-628.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Poffenberger K. L., Roizman B. A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection. J Virol. 1985 Feb;53(2):587–595. doi: 10.1128/jvi.53.2.587-595.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Preston V. G., Coates J. A., Rixon F. J. Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA. J Virol. 1983 Mar;45(3):1056–1064. doi: 10.1128/jvi.45.3.1056-1064.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sarisky R. T., Weber P. C. Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events. J Virol. 1994 Jan;68(1):34–47. doi: 10.1128/jvi.68.1.34-47.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Severini A., Morgan A. R., Tovell D. R., Tyrrell D. L. Study of the structure of replicative intermediates of HSV-1 DNA by pulsed-field gel electrophoresis. Virology. 1994 May 1;200(2):428–435. doi: 10.1006/viro.1994.1206. [DOI] [PubMed] [Google Scholar]
  33. Shao L., Rapp L. M., Weller S. K. Herpes simplex virus 1 alkaline nuclease is required for efficient egress of capsids from the nucleus. Virology. 1993 Sep;196(1):146–162. doi: 10.1006/viro.1993.1463. [DOI] [PubMed] [Google Scholar]
  34. Sharples G. J., Lloyd R. G. Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein. J Bacteriol. 1991 Dec;173(23):7711–7715. doi: 10.1128/jb.173.23.7711-7715.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sheldrick P., Berthelot N. Inverted repetitions in the chromosome of herpes simplex virus. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 2):667–678. doi: 10.1101/sqb.1974.039.01.080. [DOI] [PubMed] [Google Scholar]
  36. Sherman G., Bachenheimer S. L. DNA processing in temperature-sensitive morphogenic mutants of HSV-1. Virology. 1987 Jun;158(2):427–430. doi: 10.1016/0042-6822(87)90214-5. [DOI] [PubMed] [Google Scholar]
  37. Smiley J. R., Fong B. S., Leung W. C. Construction of a double-jointed herpes simplex viral DNA molecule: inverted repeats are required for segment inversion, and direct repeats promote deletions. Virology. 1981 Aug;113(1):345–362. doi: 10.1016/0042-6822(81)90161-6. [DOI] [PubMed] [Google Scholar]
  38. Smiley J. R., Wagner M. J., Summers W. P., Summers W. C. Genetic and physical evidence for the polarity of transcription of the thymidine kinase gene of herpes simplex virus. Virology. 1980 Apr 15;102(1):83–93. doi: 10.1016/0042-6822(80)90072-0. [DOI] [PubMed] [Google Scholar]
  39. Spaete R. R., Frenkel N. The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell. 1982 Aug;30(1):295–304. doi: 10.1016/0092-8674(82)90035-6. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Strobel-Fidler M., Francke B. Alkaline deoxyribonuclease induced by herpes simplex virus type 1: composition and properties of the purified enzyme. Virology. 1980 Jun;103(2):493–501. doi: 10.1016/0042-6822(80)90206-8. [DOI] [PubMed] [Google Scholar]
  42. Varmuza S. L., Smiley J. R. Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences. Cell. 1985 Jul;41(3):793–802. doi: 10.1016/s0092-8674(85)80060-x. [DOI] [PubMed] [Google Scholar]
  43. Vlazny D. A., Frenkel N. Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes. Proc Natl Acad Sci U S A. 1981 Feb;78(2):742–746. doi: 10.1073/pnas.78.2.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Walboomers J. M., Schegget J. T. A new method for the isolation of herpes simplex virus type 2 DNA. Virology. 1976 Oct 1;74(1):256–258. doi: 10.1016/0042-6822(76)90151-3. [DOI] [PubMed] [Google Scholar]
  45. Weber P. C., Challberg M. D., Nelson N. J., Levine M., Glorioso J. C. Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity. Cell. 1988 Jul 29;54(3):369–381. doi: 10.1016/0092-8674(88)90200-0. [DOI] [PubMed] [Google Scholar]
  46. Weller S. K., Seghatoleslami M. R., Shao L., Rowse D., Carmichael E. P. The herpes simplex virus type 1 alkaline nuclease is not essential for viral DNA synthesis: isolation and characterization of a lacZ insertion mutant. J Gen Virol. 1990 Dec;71(Pt 12):2941–2952. doi: 10.1099/0022-1317-71-12-2941. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Zhang X., Efstathiou S., Simmons A. Identification of novel herpes simplex virus replicative intermediates by field inversion gel electrophoresis: implications for viral DNA amplification strategies. Virology. 1994 Aug 1;202(2):530–539. doi: 10.1006/viro.1994.1375. [DOI] [PubMed] [Google Scholar]
  49. al-Kobaisi M. F., Rixon F. J., McDougall I., Preston V. G. The herpes simplex virus UL33 gene product is required for the assembly of full capsids. Virology. 1991 Jan;180(1):380–388. doi: 10.1016/0042-6822(91)90043-b. [DOI] [PubMed] [Google Scholar]
  50. de Massy B., Weisberg R. A., Studier F. W. Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA. J Mol Biol. 1987 Jan 20;193(2):359–376. doi: 10.1016/0022-2836(87)90224-5. [DOI] [PubMed] [Google Scholar]

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