Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Jul;71(7):4997–5002. doi: 10.1128/jvi.71.7.4997-5002.1997

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

G W Holzer 1, F G Falkner 1
PMCID: PMC191732  PMID: 9188564

Abstract

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Boris-Lawrie K. A., Temin H. M. Recent advances in retrovirus vector technology. Curr Opin Genet Dev. 1993 Feb;3(1):102–109. doi: 10.1016/s0959-437x(05)80349-1. [DOI] [PubMed] [Google Scholar]
  2. Chakrabarti S., Brechling K., Moss B. Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol Cell Biol. 1985 Dec;5(12):3403–3409. doi: 10.1128/mcb.5.12.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Condit R. C., Motyczka A. Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology. 1981 Aug;113(1):224–241. doi: 10.1016/0042-6822(81)90150-1. [DOI] [PubMed] [Google Scholar]
  4. Condit R. C., Niles E. G. Orthopoxvirus genetics. Curr Top Microbiol Immunol. 1990;163:1–39. doi: 10.1007/978-3-642-75605-4_1. [DOI] [PubMed] [Google Scholar]
  5. Falkner F. G., Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol. 1988 Jun;62(6):1849–1854. doi: 10.1128/jvi.62.6.1849-1854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Forrester A., Farrell H., Wilkinson G., Kaye J., Davis-Poynter N., Minson T. Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. J Virol. 1992 Jan;66(1):341–348. doi: 10.1128/jvi.66.1.341-348.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  8. Herlitschka S. E., Falkner F. G., Schlokat U., Dorner F. Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker. Protein Expr Purif. 1996 Nov;8(3):358–364. doi: 10.1006/prep.1996.0111. [DOI] [PubMed] [Google Scholar]
  9. JOKLIK W. K. The purification fo four strains of poxvirus. Virology. 1962 Sep;18:9–18. doi: 10.1016/0042-6822(62)90172-1. [DOI] [PubMed] [Google Scholar]
  10. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [DOI] [PubMed] [Google Scholar]
  11. Ligas M. W., Johnson D. C. A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells. J Virol. 1988 May;62(5):1486–1494. doi: 10.1128/jvi.62.5.1486-1494.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McFadden G., Dales S. Biogenesis of poxviruses: preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis. Virology. 1980 May;103(1):68–79. doi: 10.1016/0042-6822(80)90126-9. [DOI] [PubMed] [Google Scholar]
  13. Millns A. K., Carpenter M. S., DeLange A. M. The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication. Virology. 1994 Feb;198(2):504–513. doi: 10.1006/viro.1994.1061. [DOI] [PubMed] [Google Scholar]
  14. Moss B. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11341–11348. doi: 10.1073/pnas.93.21.11341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Paoletti E. Applications of pox virus vectors to vaccination: an update. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11349–11353. doi: 10.1073/pnas.93.21.11349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Randrianarison-Jewtoukoff V., Perricaudet M. Recombinant adenoviruses as vaccines. Biologicals. 1995 Jun;23(2):145–157. doi: 10.1006/biol.1995.0025. [DOI] [PubMed] [Google Scholar]
  17. Scheiflinger F., Falkner F. G., Dorner F. Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain. Arch Virol. 1996;141(3-4):663–669. doi: 10.1007/BF01718324. [DOI] [PubMed] [Google Scholar]
  18. Stuart D. T., Upton C., Higman M. A., Niles E. G., McFadden G. A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. J Virol. 1993 May;67(5):2503–2512. doi: 10.1128/jvi.67.5.2503-2512.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sutter G., Ramsey-Ewing A., Rosales R., Moss B. Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J Virol. 1994 Jul;68(7):4109–4116. doi: 10.1128/jvi.68.7.4109-4116.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Upton C., Stuart D. T., McFadden G. Identification of a poxvirus gene encoding a uracil DNA glycosylase. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4518–4522. doi: 10.1073/pnas.90.10.4518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zhang Y. F., Moss B. Inducer-dependent conditional-lethal mutant animal viruses. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1511–1515. doi: 10.1073/pnas.88.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES