Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Dec;70(12):8801–8812. doi: 10.1128/jvi.70.12.8801-8812.1996

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

D W Martin 1, P C Weber 1
PMCID: PMC190977  PMID: 8971009

Abstract

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.

Full Text

The Full Text of this article is available as a PDF (443.3 KB).

Selected References

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

  1. Bataille D., Epstein A. Herpes simplex virus replicative concatemers contain L components in inverted orientation. Virology. 1994 Sep;203(2):384–388. doi: 10.1006/viro.1994.1498. [DOI] [PubMed] [Google Scholar]
  2. Bruckner R. C., Dutch R. E., Zemelman B. V., Mocarski E. S., Lehman I. R. Recombination in vitro between herpes simplex virus type 1 a sequences. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10950–10954. doi: 10.1073/pnas.89.22.10950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buller R. M., Chakrabarti S., Cooper J. A., Twardzik D. R., Moss B. Deletion of the vaccinia virus growth factor gene reduces virus virulence. J Virol. 1988 Mar;62(3):866–874. doi: 10.1128/jvi.62.3.866-874.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou J., Roizman B. Isomerization of herpes simplex virus 1 genome: identification of the cis-acting and recombination sites within the domain of the a sequence. Cell. 1985 Jul;41(3):803–811. doi: 10.1016/s0092-8674(85)80061-1. [DOI] [PubMed] [Google Scholar]
  5. Davison A. J., Wilkie N. M. Inversion of the two segments of the herpes simplex virus genome in intertypic recombinants. J Gen Virol. 1983 Jan;64(Pt 1):1–18. doi: 10.1099/0022-1317-64-1-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Goldin A. L., Sandri-Goldin R. M., Levine M., Glorioso J. C. Cloning of herpes simplex virus type 1 sequences representing the whole genome. J Virol. 1981 Apr;38(1):50–58. doi: 10.1128/jvi.38.1.50-58.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Jenkins F. J., Casadaban M. J., Roizman B. Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4773–4777. doi: 10.1073/pnas.82.14.4773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jenkins F. J., Roizman B. Herpes simplex virus 1 recombinants with noninverting genomes frozen in different isomeric arrangements are capable of independent replication. J Virol. 1986 Aug;59(2):494–499. doi: 10.1128/jvi.59.2.494-499.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones N., Shenk T. Isolation of deletion and substitution mutants of adenovirus type 5. Cell. 1978 Jan;13(1):181–188. doi: 10.1016/0092-8674(78)90148-4. [DOI] [PubMed] [Google Scholar]
  16. Kapoor Q. S., Chinnadurai G. Method for introducing site-specific mutations into adenovirus 2 genome: construction of a small deletion mutant in VA-RNAI gene. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2184–2188. doi: 10.1073/pnas.78.4.2184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Knipe D. M., Ruyechan W. T., Honess R. W., Roizman B. Molecular genetics of herpes simplex virus: the terminal a sequences of the L and S components are obligatorily identical and constitute a part of a structural gene mapping predominantly in the S component. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4534–4538. doi: 10.1073/pnas.76.9.4534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Longnecker R., Roizman B. Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene. J Virol. 1986 May;58(2):583–591. doi: 10.1128/jvi.58.2.583-591.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McKnight S. L., Gavis E. R. Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro. Nucleic Acids Res. 1980 Dec 20;8(24):5931–5948. doi: 10.1093/nar/8.24.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mocarski E. S., Post L. E., Roizman B. Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions. Cell. 1980 Nov;22(1 Pt 1):243–255. doi: 10.1016/0092-8674(80)90172-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mocarski E. S., Roizman B. Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA. Cell. 1982 Nov;31(1):89–97. doi: 10.1016/0092-8674(82)90408-1. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Poffenberger K. L., Tabares E., Roizman B. Characterization of a viable, noninverting herpes simplex virus 1 genome derived by insertion and deletion of sequences at the junction of components L and S. Proc Natl Acad Sci U S A. 1983 May;80(9):2690–2694. doi: 10.1073/pnas.80.9.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pogue-Geile K. L., Lee G. T., Spear P. G. Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component. J Virol. 1985 Feb;53(2):456–461. doi: 10.1128/jvi.53.2.456-461.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pogue-Geile K. L., Spear P. G. Enhanced rate of conversion or recombination of markers within a region of unique sequence in the herpes simplex virus genome. J Virol. 1986 May;58(2):704–708. doi: 10.1128/jvi.58.2.704-708.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Post L. E., Roizman B. A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth. Cell. 1981 Jul;25(1):227–232. doi: 10.1016/0092-8674(81)90247-6. [DOI] [PubMed] [Google Scholar]
  28. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Smiley J. R. Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus. Nature. 1980 May 29;285(5763):333–335. doi: 10.1038/285333a0. [DOI] [PubMed] [Google Scholar]
  32. Smiley J. R., Duncan J., Howes M. Sequence requirements for DNA rearrangements induced by the terminal repeat of herpes simplex virus type 1 KOS DNA. J Virol. 1990 Oct;64(10):5036–5050. doi: 10.1128/jvi.64.10.5036-5050.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Smiley J. R., Lavery C., Howes M. The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome. J Virol. 1992 Dec;66(12):7505–7510. doi: 10.1128/jvi.66.12.7505-7510.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Umene K. Intermolecular recombination of the herpes simplex virus type 1 genome analysed using two strains differing in restriction enzyme cleavage sites. J Gen Virol. 1985 Dec;66(Pt 12):2659–2670. doi: 10.1099/0022-1317-66-12-2659. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Varmuza S. L., Smiley J. R. Unstable heterozygosity in a diploid region of herpes simplex virus DNA. J Virol. 1984 Feb;49(2):356–362. doi: 10.1128/jvi.49.2.356-362.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Weber P. C., Kenny J. J., Wigdahl B. Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0. J Gen Virol. 1992 Nov;73(Pt 11):2955–2961. doi: 10.1099/0022-1317-73-11-2955. [DOI] [PubMed] [Google Scholar]
  40. Weber P. C., Levine M., Glorioso J. C. Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes. J Virol. 1990 Jan;64(1):300–306. doi: 10.1128/jvi.64.1.300-306.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wohlrab F., Chatterjee S., Wells R. D. The herpes simplex virus 1 segment inversion site is specifically cleaved by a virus-induced nuclear endonuclease. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6432–6436. doi: 10.1073/pnas.88.15.6432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wohlrab F., McLean M. J., Wells R. D. The segment inversion site of herpes simplex virus type 1 adopts a novel DNA structure. J Biol Chem. 1987 May 5;262(13):6407–6416. [PubMed] [Google Scholar]

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

RESOURCES