1887

Abstract

In the late stages of an entomopoxvirus infectissson, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the con-struction of a genetically modified entomopoxvirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique HI site of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The recombinant virus was isolated from wild-type virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS–PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was able to replicate to high titres (10 p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication . Moderate levels of CAT were synthesized in recombinant virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified virus.

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1995-01-01
2021-10-18
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References

  1. Bilimoria S. L., Arif B. M. 1979; Subunit protein and alkaline protease of entomopoxvirus spheroids. Virology 96:596–603
    [Google Scholar]
  2. Blissard G. W., Rohrmann G. F. 1990; Baculovirus diversity and molecular biology. Annual Review of Entomology 35:127–155
    [Google Scholar]
  3. Davison A. J., Moss B. 1989; Structure of vaccinia virus late promoters. Journal of Molecular Biology 210:749–769
    [Google Scholar]
  4. Francki R. I. B., Fauquet C. M., Knudson D. L., Brown F. 1991; Classification and Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses. Archives of Virology Supplementum 2
    [Google Scholar]
  5. Goodwin R. H., Adams J. R., Shapiro M. 1990; Replication of the entomopoxvirus from Amsacta moorei in serum-free cultures of a gypsy moth cell line. Journal of Invertebrate Pathology 56:190–205
    [Google Scholar]
  6. Gorman C. M., Moffat L. F., Howard B. H. 1982; Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Molecular and Cellular Biology 2:1044–1051
    [Google Scholar]
  7. Granados R. R. 1981; Entomopoxvirus infections in insects. In Pathogenesis of Invertebrate Microbial Diseases102–106 Davidson E. W. New Jersey: Allanheld Totowa;
    [Google Scholar]
  8. Granados R. R., Roberts D. W. 1970; Electron microscopy of a pox-like virus infecting an invertebrate host. Virology 40:230–243
    [Google Scholar]
  9. Gruidl M. E., Hall R. L., Moyer R. W. 1992; Mapping and molecular characterization of a functional thymidine kinase from Amsacta moorei entomopoxvirus. Virology 187:507–516
    [Google Scholar]
  10. Hall R. L., Hink W. F. 1990; Physical mapping and field inversion gel electrophoresis of Amsacta moorei entomopoxvirus DNA. Archives of Virology 110:77–90
    [Google Scholar]
  11. Hall R. L., Moyer R. W. 1991; Identification, cloning and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames. Virology 65:6516–6527
    [Google Scholar]
  12. Hall R. L., Moyer R. W. 1993; Identification of an Amsacta spheroidin-like protein within the occlusion bodies of Choristoneura entomopoxviruses. Virology 192:179–187
    [Google Scholar]
  13. Innis M. A., Gelfand D. H., Sninsky J. J., White T. J. 1990 PCR Protocols, a Guide to Methods and Applications San Diego: Academic Press;
    [Google Scholar]
  14. King L. A., Possee R. D. 1992 The Baculovirus Expression System, a Laboratory Guide. London: Chapman and Hall;
    [Google Scholar]
  15. Langridge W. H. R., Roberts D. W. 1982; Structural proteins of Amsacta moorei, Euxoa auxillaris, and Melanoplus sanguinipes entomopoxviruses. Journal of Invertebrate Pathology 39:346–353
    [Google Scholar]
  16. Langridge W. H. R., Bozarth R. F., Roberts D. W. 1977; The base composition of entomopoxvirus DNA. Virology 76:616–620
    [Google Scholar]
  17. Mackett M. 1992; Vaccinia virus vectors. In Transgenics155–186 Murray J. A. H. London: John Wiley;
    [Google Scholar]
  18. Mackett M., Smith G. L., Moss B. 1985; The construction and characterisation of vaccinia virus recombinants expressing foreign genes. In DNA Cloning: A Practical Approach191–211 Glover D. M. Oxford: IRL Press;
    [Google Scholar]
  19. Marlow S. A., Palmer C. P., King L. A. 1992; The cytopathic effects of Amsacta moorei entomopoxvirus infection on the cyto- skeleton of Estigmene acrea cells. Virus Research 26:41–55
    [Google Scholar]
  20. Marlow S. A., Palmer C. P., King L. A. 1993; Replication and morphogenesis of Amsacta moorei entomopoxvirus in cultured cells of Estigmene acrea (salt marsh caterpillar). Journal of General Virology 74:1457–1461
    [Google Scholar]
  21. Moss B. 1990; Poxviridae and their replication. In Fields’ Virology, 2.2079–2111 Fields B. N., Knipe D. M., Chanock R. M., Hirsch M. S., Melnick J. L., Monath T. P., Roizman B. New York: Raven Press;
    [Google Scholar]
  22. O’Reilly D. R., Miller L. K., Luckow V. A. 1992; Baculovirus Expression Vectors. A Laboratory Manual. New York: Freeman W. H.
    [Google Scholar]
  23. Palmer C. P. 1993 Molecular biology of entomopoxviruses PhD thesis, Oxford Brookes University;Oxford, UK
    [Google Scholar]
  24. Patel D. D., Pickup D. J. 1987; Messenger RNAs of a strongly expressed late gene of cowpox virus contains 5′-terminal poly(A) sequences. EMBO Journal 63787–3794
    [Google Scholar]
  25. Richardson C. D., Banville M., Lalumlere M., Vialard J., Meighen E. A. 1992; Bacterial luciferase produced with rapidscreening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirology 34:213–227
    [Google Scholar]
  26. Rosel J. L., Earl P. L., Weir J. P., Moss B. 1986; Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII ‘H’ genome fragment. Journal of Virologv 60:436–449
    [Google Scholar]
  27. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of SciencesUSA 74:5463–5467
    [Google Scholar]
  29. Smith G. E., Fraser M. J., Summers M. D. 1983; Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: deletion mutants within the polyhedrin gene. Journal of Virology 46:584–593
    [Google Scholar]
  30. Smith G. E., Ju G., Ericson B. L., Moschera J., Lahm H., Chizzonite R., Summers M. D. 1983; Modification and secretion of human interleukin-2 produced in insect cells by a baculovirus vector. Proceedings of the National Academy of SciencesUSA 82:8404–8408
    [Google Scholar]
  31. Southern E. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  32. Woods S. A., Street D. A., Henry J. E. 1992; Temporal patterns of mortality from an entomopoxvirus and strategies for control of the migratory grasshopper. (Melanoplus sanguinipes F.). Journal of Invertebrate Pathology 60:33–39
    [Google Scholar]
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