Skip to main content
SpringerLink
Log in

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of3H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ball, L. A. 1987. High frequency homologous recombination in vaccinia virus DNA. J. Virol. 61: 1788–1795.

    PubMed  CAS  Google Scholar 

  2. Belle Isle, H., S. Venkatesan and B. Moss. 1981. Cell free translation of early and late mRNAs selected by hybridzation to cloned DNA fragments derived from the left 4 to 72 million daltons of the vaccinia virus genome. Virology 112: 306–317.

    Article  CAS  Google Scholar 

  3. Campbell, J. L. 1986. Eukaryotic DNA replication. Ann. Rev. Biochem. 55: 733–771.

    Article  PubMed  CAS  Google Scholar 

  4. Cochran, M.A., M. Mackett and B. Moss. 1984. Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. Proc. Natl. Acad. Sci. USA 82:19–23.

    Article  Google Scholar 

  5. Colinas, R. J., Condit, R. C. and E. Paoletti. 1990. Extrachromosomal recombination in vacccinia-infected cells requires a functional DNA polymerase at a level other than DNA replication. Virus Res. 18: 49–70.

    Article  PubMed  CAS  Google Scholar 

  6. Condit, R. C., A. Motyczka. 1981. Isolation and preliminary characterization of temperature sensitive mutants of vaccinia virus. Virology. 113: 224–241.

    Article  PubMed  CAS  Google Scholar 

  7. Condit, R. C., A. Motyczka and G. Spizz. 1983. Isolation, characterization and physical mapping of temperature sensitive mutants of vaccinia virus. Virology. 128: 429–443.

    Article  PubMed  CAS  Google Scholar 

  8. Condit, R.C. and E.G. Niles. 1990. Orthopox genetics. Curr. Topics in Microbiol. and Immunol. 163: 1–39.

    CAS  Google Scholar 

  9. Dales, S., Milanovitch, V., Pogo, B. G. T., Weintraub, S., Huima, T., Wilton, S., and G. McFadden. 1978. Biogenesis of vaccinia: isolation of conditional lethal mutants and electron microscopic characterization of their phenotypically expressed defects. Virology. 84: 403–428.

    Article  PubMed  CAS  Google Scholar 

  10. DeLange, A. M., and G. McFadden. 1986. Sequence non-specific replication of transfected plasmid DNA in poxvirus-infected cells. Proc. Natl. Acad. Sci. USA. 83: 614–618.

    Article  PubMed  CAS  Google Scholar 

  11. Elroy-Stein, O., T. R. Fuerst and B. Moss. 1989. Cap-independent translation of mRNA conferred by EMC virus 5′ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl. Acad. Sci. USA. 86: 6126–6130.

    Article  PubMed  CAS  Google Scholar 

  12. Ensinger, M. 1982. Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J. Virol. 43: 778–790.

    PubMed  CAS  Google Scholar 

  13. Ensinger, M. and M. Rovinsky. 1983. Marker rescue of temperature sensitive mutation of vaccinia virus WR: Correlation of genetic and physical maps. J. Virology. 48: 419–428.

    PubMed  CAS  Google Scholar 

  14. Ensinger, M. 1987. Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutation in a 135–000-Mr subunit of the virion-associated DNA-dependant RNA polymerase. J. Virol. 61: 1842–1850.

    PubMed  CAS  Google Scholar 

  15. Evans, D.H., Stuart, D. and G. McFadden. 1988. High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells. J. Virol. 62: 367–375.

    PubMed  CAS  Google Scholar 

  16. Evans, E., and P. Traktman, 1987. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication. J. Virol. 61: 3152–3162.

    PubMed  CAS  Google Scholar 

  17. Fisher, C., Parks, R. J. Lauzon, M. L. and D. H. Evans. 1991. Heteroduplex formation is associated with replication and recombination in poxvirus- infected cells. Genetics. 129: 7–18.

    PubMed  CAS  Google Scholar 

  18. Goebel, S. J., G. P. Johnson, M. E. Perkus, S. W. David, J. P. Winslow and E. Paoletti. 1990. The complete DNA sequence of vaccinia virus. Virology. 179: 247–266.

    Article  PubMed  CAS  Google Scholar 

  19. Heyer, W., M. R. S. Rao, L. F. Erdile, T. J. Kelly and R. D. Kolodner. An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A. EMBO J. 9: 2321–2329.

  20. Ish-Horowicz, D. and J. F. Burke. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9: 2989–2998.

    PubMed  CAS  Google Scholar 

  21. Joklik, W. K. 1962. The purification and characterization of highly purified radioactively labeled poxvirus. Biochim. Biophys. Acta. 61: 290–301.

    PubMed  CAS  Google Scholar 

  22. Lindenbaum, J.O., J. Field and J. Hurwitz. 1986. The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed, DNA templates. J. Biol. Chem. 261: 10218–10227.

    PubMed  CAS  Google Scholar 

  23. Maxam, A.M. and W. Gilbert. 1980. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65:497–559.

    Article  Google Scholar 

  24. McFadden, G. and S. Dales. 1980. Biogenesis of Poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis. Virology. 103: 68–79

    Article  PubMed  CAS  Google Scholar 

  25. Merchlinsky, M. 1989. Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus. J. Virol. 63: 2030–2035.

    PubMed  CAS  Google Scholar 

  26. Niles, E. G., R. C. Condit, P. Caro, K. Davidson, L. Matusick and J. Seto. 1986. Nucleotide sequence and genetic map of the 16 kb vaccinia virus Hind III D fragment. J. Virol. 153: 96–112.

    Article  CAS  Google Scholar 

  27. Parks, R.J. and D.H. Evans. 1991. Effect of marker distance and orientation on recombinant formation in poxivrus-infected cells. J. Virol. 65:1263–1272.

    PubMed  CAS  Google Scholar 

  28. Prives, C. 1990. The replication functions of SV-40 T-antigen are regulated by phosphorylation. Cell 61: 735–738.

    Article  PubMed  CAS  Google Scholar 

  29. Rempel, R. E., M. K. Anderson, E. Evans, and P. Traktman. 1990. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication. J. Virol. 64: 574–583.a

    PubMed  CAS  Google Scholar 

  30. Rempel, R.E., and P. Traktman. 1992. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins. J. Virol. 66: 4413–4426.

    PubMed  CAS  Google Scholar 

  31. Roseman, N.A. and D.E. Hruby. 1987. Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication. J. Virol. 61: 1398–1406.

    PubMed  CAS  Google Scholar 

  32. Salah-ud-Din, Brill, S.J., Fairman, M.P. and B. Stillman. 1990. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes and Development 4: 968–977.

    Google Scholar 

  33. Seto, J., Celenza, L. M., Condit, R. C. and E. Niles. 1987. Genetic map of the vaccinia virus HindIII D fragment. Virology. 160: 110–119.

    Article  PubMed  CAS  Google Scholar 

  34. Spyropoulos D. D., Roberts, B. E., Panicali, D. L. and L. K. Cohen. 1988. Delineation of the viral products of recombination in vaccinia virus-infected cells. J. Virol. 62: 1046–1054.

    PubMed  CAS  Google Scholar 

  35. Taddie, J. A. and P. Traktman. 1991. Genetic characterization of the vaccinia virus DNA polymerase: Identification of point mutations conferring altered drug sensitivities and reeduced fidelity. J. Virol. 65: 869–879.

    PubMed  CAS  Google Scholar 

  36. Traktman P. 1990. The enzymology of poxvirus DNA replication. Current Topics in Micro. and Immunol. 163: 93–123.

    CAS  Google Scholar 

  37. Traktman, P., 1991. Molecular genetic, and biochemical analysis of poxvirus DNA replication. Sem. Virol. 2: 291–304.

    CAS  Google Scholar 

  38. Traktman, P., M. K. Anderson and R. E. Rempel. 1989. Vaccinia virus encodes an essential gene with strong homology to protein kinases. J. Biol. Chem. 264: 21458–21461.

    PubMed  CAS  Google Scholar 

  39. Traktman, P., Kelvin, M. and S. Pacheco. 1989. Molecular genetic analysis of vaccinia virus DNA polymerase mutants. J. Virol. 63: 841–846.

    PubMed  CAS  Google Scholar 

  40. Vogelstein, B. and D. Gillespie. 1979. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA. 76: 615–619.

    Article  PubMed  CAS  Google Scholar 

  41. Wang, T.S.-F. 1991. Eukaryotic DNA polymerases. Ann. Rev. Biochem. 60: 513–553.

    Article  PubMed  CAS  Google Scholar 

  42. West, S. C. and P. Howard-Flanders 1984. Duplex-duplex interactions catalyzed by Rec A protein allow strand exchanges to pass double-strand breaks in DNA. Cell 37: 683–691.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Elizabeth Evans

    Present address: The Rockefeller Foundation, 1133 Ave. of the Americas, 10036, New York, NY, USA

Authors and Affiliations

  1. Department of Cell Biology & Anatomy, Cornell University Medical College, 10021, NY, New York, USA

    Elizabeth Evans & Paula Traktman

  2. Department of Microbiology, Cornell University Medical College, 10021, New York, NY, USA

    Paula Traktman

Authors
  1. Elizabeth Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paula Traktman
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Evans, E., Traktman, P. Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene. Chromosoma 102 (Suppl 1), S72–S82 (1992). https://doi.org/10.1007/BF02451789

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02451789

Keywords

Search

Navigation