Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1985 Aug;55(2):338–346. doi: 10.1128/jvi.55.2.338-346.1985

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

A E Sears, I W Halliburton, B Meignier, S Silver, B Roizman
PMCID: PMC254938  PMID: 2991560

Abstract

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.

Full text

PDF
340

Images in this article

340Image
on p.340
341Image
on p.341
341Image
on p.341
344Image
on p.344

Selected References

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

  1. Cohen J. C., Henry B. E., Randall C. C., O'Callaghan D. J. Ribonucleotide reductase activity in hydroxyurea-resistant herpesvirus replication. Proc Soc Exp Biol Med. 1977 Jul;155(3):395–399. doi: 10.3181/00379727-155-39815. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Dixon R. A., Schaffer P. A. Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175. J Virol. 1980 Oct;36(1):189–203. doi: 10.1128/jvi.36.1.189-203.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ejercito P. M., Kieff E. D., Roizman B. Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol. 1968 May;2(3):357–364. doi: 10.1099/0022-1317-2-3-357. [DOI] [PubMed] [Google Scholar]
  5. Hall L. M., Draper K. G., Frink R. J., Costa R. H., Wagner E. K. Herpes simplex virus mRNA species mapping in EcoRI fragment I. J Virol. 1982 Aug;43(2):594–607. doi: 10.1128/jvi.43.2.594-607.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hoffmann P. J., Cheng Y. C. DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity. J Virol. 1979 Nov;32(2):449–457. doi: 10.1128/jvi.32.2.449-457.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Holland L. E., Anderson K. P., Shipman C., Jr, Wagner E. K. Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species. Virology. 1980 Feb;101(1):10–24. doi: 10.1016/0042-6822(80)90479-1. [DOI] [PubMed] [Google Scholar]
  9. Honess R. W., Roizman B. Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J Virol. 1973 Dec;12(6):1347–1365. doi: 10.1128/jvi.12.6.1347-1365.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Honess R. W., Roizman B. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol. 1974 Jul;14(1):8–19. doi: 10.1128/jvi.14.1.8-19.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honess R. W., Roizman B. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1276–1280. doi: 10.1073/pnas.72.4.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honess R. W., Watson D. H. Herpes simplex virus resistance and sensitivity to phosphonoacetic acid. J Virol. 1977 Feb;21(2):584–600. doi: 10.1128/jvi.21.2.584-600.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huszar D., Bacchetti S. Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells. J Virol. 1981 Feb;37(2):580–588. doi: 10.1128/jvi.37.2.580-588.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jamieson A. T., Gentry G. A., Subak-Sharpe J. H. Induction of both thymidine and deoxycytidine kinase activity by herpes viruses. J Gen Virol. 1974 Sep;24(3):465–480. doi: 10.1099/0022-1317-24-3-465. [DOI] [PubMed] [Google Scholar]
  15. Jamieson A. T., Subak-Sharpe J. H. Biochemical studies on the herpes simplex virus-specified deoxypyrimidine kinase activity. J Gen Virol. 1974 Sep;24(3):481–492. doi: 10.1099/0022-1317-24-3-481. [DOI] [PubMed] [Google Scholar]
  16. Jones P. C., Hayward G. S., Roizman B. Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA. J Virol. 1977 Jan;21(1):268–276. doi: 10.1128/jvi.21.1.268-276.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones P. C., Roizman B. Regulation of herpesvirus macromolecular synthesis. VIII. The transcription program consists of three phases during which both extent of transcription and accumulation of RNA in the cytoplasm are regulated. J Virol. 1979 Aug;31(2):299–314. doi: 10.1128/jvi.31.2.299-314.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. KIT S., DUBBS D. R. Acquisition of thymidine kinase activity by herpes simplex-infected mouse fibroblast cells. Biochem Biophys Res Commun. 1963 Apr 2;11:55–59. doi: 10.1016/0006-291x(63)90027-5. [DOI] [PubMed] [Google Scholar]
  19. Knipe D. M., Ruyechan W. T., Roizman B., Halliburton I. W. Molecular genetics of herpes simplex virus: demonstration of regions of obligatory and nonobligatory identity within diploid regions of the genome by sequence replacement and insertion. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3896–3900. doi: 10.1073/pnas.75.8.3896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kousoulas K. G., Pellett P. E., Pereira L., Roizman B. Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene. Virology. 1984 Jun;135(2):379–394. doi: 10.1016/0042-6822(84)90194-6. [DOI] [PubMed] [Google Scholar]
  21. Mackem S., Roizman B. Regulation of herpesvirus macromolecular synthesis: temporal order of transcription of alpha genes is not dependent on the stringency of inhibition of protein synthesis. J Virol. 1981 Oct;40(1):319–322. doi: 10.1128/jvi.40.1.319-322.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marsden H. S., Crombie I. K., Subak-Sharpe J. H. Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17. J Gen Virol. 1976 Jun;31(3):347–372. doi: 10.1099/0022-1317-31-3-347. [DOI] [PubMed] [Google Scholar]
  23. Morse L. S., Pereira L., Roizman B., Schaffer P. A. Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants. J Virol. 1978 May;26(2):389–410. doi: 10.1128/jvi.26.2.389-410.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Palmiter R. D., Chen H. Y., Brinster R. L. Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell. 1982 Jun;29(2):701–710. doi: 10.1016/0092-8674(82)90186-6. [DOI] [PubMed] [Google Scholar]
  25. Pereira L., Wolff M. H., Fenwick M., Roizman B. Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells. Virology. 1977 Apr;77(2):733–749. doi: 10.1016/0042-6822(77)90495-0. [DOI] [PubMed] [Google Scholar]
  26. Post L. E., Mackem S., Roizman B. Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell. 1981 May;24(2):555–565. doi: 10.1016/0092-8674(81)90346-9. [DOI] [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. Powell K. L., Purifoy D. J. Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. J Virol. 1977 Nov;24(2):618–626. doi: 10.1128/jvi.24.2.618-626.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Preston C. M., Cordingley M. G., Stow N. D. Analysis of DNA sequences which regulate the transcription of a herpes simplex virus immediate early gene. J Virol. 1984 Jun;50(3):708–716. doi: 10.1128/jvi.50.3.708-716.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Preston C. M., Cordingley M. G. mRNA- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes. J Virol. 1982 Aug;43(2):386–394. doi: 10.1128/jvi.43.2.386-394.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Preston V. G., Davison A. J., Marsden H. S., Timbury M. C., Subak-Sharpe J. H., Wilkie N. M. Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides. J Virol. 1978 Nov;28(2):499–517. doi: 10.1128/jvi.28.2.499-517.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Purifoy D. J., Powell K. L. Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants. J Virol. 1977 Nov;24(2):470–477. doi: 10.1128/jvi.24.2.470-477.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roizman B., Warren J., Thuning C. A., Fanshaw M. S., Norrild B., Meignier B. Application of molecular genetics to the design of live herpes simplex virus vaccines. Dev Biol Stand. 1982;52:287–304. [PubMed] [Google Scholar]
  34. Silver S., Roizman B. gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes. Mol Cell Biol. 1985 Mar;5(3):518–528. doi: 10.1128/mcb.5.3.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Summers W. C., Summers W. P. [125I]deoxycytidine used in a rapid, sensitive, and specific assay for herpes simplex virus type 1 thymidine kinase. J Virol. 1977 Oct;24(1):314–318. doi: 10.1128/jvi.24.1.314-318.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Topp W. C. Normal rat cell lines deficient in nuclear thymidine kinase. Virology. 1981 Aug;113(1):408–411. doi: 10.1016/0042-6822(81)90168-9. [DOI] [PubMed] [Google Scholar]
  37. Watson R. J., Clements J. B. A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature. 1980 May 29;285(5763):329–330. doi: 10.1038/285329a0. [DOI] [PubMed] [Google Scholar]
  38. Wilcox K. W., Kohn A., Sklyanskaya E., Roizman B. Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. J Virol. 1980 Jan;33(1):167–182. doi: 10.1128/jvi.33.1.167-182.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES