1887

Abstract

The -endotoxin gene from subsp. HD-73 was inserted into nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the - endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a poly- hedrin-positive virus. Analysis of infected cell extracts showed that the -endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the -endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD value. The polyhedrin-positive virus had an LD value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-7-1535
1990-07-01
2024-12-13
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/7/JV0710071535.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-7-1535&mimeType=html&fmt=ahah

References

  1. Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. 1985; Characterized full length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta . Gene 36:289–300
    [Google Scholar]
  2. Bishop D. H. L., Entwistle P. F., Cameron I. R., Allen C. J., Possee R. D. 1988; Field trials with genetically engineered baculovirus insecticides. In The Release of Genetically Engineered Micro-organisms pp. 143–179 Sussman M., Collins C. H., Skinner F. A., Stewart-Tull D. E. Edited by New York & London: Academic Press;
    [Google Scholar]
  3. Brown M., Faulkner P. 1977; A plaque assay for nuclear polyhedrosis virus using a solid overlay. Journal of General Virology 36:361–364
    [Google Scholar]
  4. Burges H. D. editor 1981 Microbial Control of Pests and Plant Diseases 1970-1980 New York & London: Academic Press;
    [Google Scholar]
  5. Cook R. F., Avery R. J., Dimmock N. J. 1979; Infection of chicken erythrocytes with influenza and other viruses. Infection and Immunity 25:396–402
    [Google Scholar]
  6. Cunningham J. C. 1982; Field trials with baculoviruses: control of forest insect pests. In Microbial and Viral Insecticides pp. 335–386 Kurstak E. Edited by New York: Marcel Dekker;
    [Google Scholar]
  7. Cunningham J. C. 1988; Baculoviruses: their status compared to Bacillus thuringiensis as microbial insecticides. Outlook on Agriculture 17:10–17
    [Google Scholar]
  8. Entwistle P. F., Evans H. F. 1985; Viral control. In Comprehensive Insect Physiology, Biochemistry and Pharmacology pp. 347–412 Kerkut G. A., Gilbert L. I. Edited by Oxford: Pergamon Press;
    [Google Scholar]
  9. Entwistle P. F., Cory J. S., Doyle C. 1988; An overview of insect baculovirus ecology as a background to field release of a genetically manipulated nuclear polyhedrosis virus. In Risk Assessment for Deliberate Releases pp. 72–80 Klingmüller W. Edited by Berlin: Springer-Verlag;
    [Google Scholar]
  10. Evans H. F., Entwistle P. F. 1987; Viral diseases. In Epizootiology of Insect Diseases pp. 257–322 Fuxa J. R., Tanada Y. Edited by New York: John Wiley;
    [Google Scholar]
  11. Goldberg L. J., Margalit J. 1977; A Bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia uniguiculata, Culex univitattus, Aedes aegypti and Culex pipiens . Mosquito News 37:355–358
    [Google Scholar]
  12. Gonzalez J. M. Jr Dulmage H. T., Carlton B. C. 1981; Correlation between specific plasmids and delta-endotoxin production in Bacillus thuringiensis . Plasmid 5:351–365
    [Google Scholar]
  13. Gonzalez J. M. Jr Brown B. J., Carlton B. C. 1982; Transfer of Bacillus thuringiensis plasmids coding for δ-endotoxin among strains of B. thuringiensis and B. cereus . Proceedings of the National Academy of Sciences, U.S.A 79:6951–6955
    [Google Scholar]
  14. Hall I. M., Arakawa K. Y., Dulmage H. T., Correa J. A. 1977; The pathogenicity of strains of Bacillus thuringiensis to larvae of Aedes and Culex mosquitoes. Mosquito News 37:246–251
    [Google Scholar]
  15. Harrap K. A., Payne C. C., Robertson J. S. 1977; The properties of three baculoviruses from closely related hosts. Virology 79:14–31
    [Google Scholar]
  16. Heimpel A. M., Angus T. A. 1959; The site of action of crystalliferous bacteria in Lepidoptera larvae. Journal of Insect Pathology 1:152–170
    [Google Scholar]
  17. Held G. A., Bulla L. A. Jr Ferrari E., Hoch J. A., Aronson A. I., Minnich S. A. 1982; Cloning and localization of the lepidopteranprotoxin gene of Bacillus thuringiensis subsp. kurstaki . Proceedings of the National Academy of Sciences, U.S.A 79:6065–6069
    [Google Scholar]
  18. Hoffman C., Luthy P., Hutter R., Pliska V. 1988a; Binding of the delta endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae). European Journal of Biochemistry 173:85–91
    [Google Scholar]
  19. Hoffman C., Vanderbruggen H., Hofte H., Van Rie J., Jansens S., Van Mellaert H. 1988b; Specificity of Bacillus thuringiensis δ-endotoxins is correlated with the presence of high-affinity binding sites in the brush-border membrane of target insect midguts. Proceedings of the National Academy of Sciences, U.S.A 85:7844–7848
    [Google Scholar]
  20. Hofte H., Whiteley H. R. 1989; Insecticidal proteins of Bacillus thuringiensis . Microbiological Reviews 53:242–255
    [Google Scholar]
  21. Huber J. 1986; Use of baculoviruses in pest management programs. In The Biology of Baculoviruses. II. Practical Applications for Insect Control pp. 181–190 Granados R. R., Federici B. A. Edited by Boca Raton: CRC Press;
    [Google Scholar]
  22. Hunter F. R., Crook N. E., Entwistle P. F. 1984; Viruses as pathogens for the control of insects. In Microbial Methods for Environmental Biotechnology pp. 323–347 Grainger J. M., Lynch J. M. Edited by New York & London: Academic Press;
    [Google Scholar]
  23. Kelly D. C. 1982; Baculovirus replication. Journal of General Virology 63:1–13
    [Google Scholar]
  24. Kelly D. C., Lescott T. 1981; Baculovirus replication: protein synthesis in Spodoptera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Microbiologica 4:35–47
    [Google Scholar]
  25. Knowles B. H., Ellar D. J. 1987; Colloid-osmotic lysis is a general feature of the mechanism of Bacillus thuringiensis δ-endotoxins with different insect specificities. Biochimica et biophysica acta 924:509–518
    [Google Scholar]
  26. Krieg A., Huger A., Langenbruch G., Schnetter W. 1983; Bacillus thuringiensis var. tenebrionis: a new pathotype effective against larvae of Coleoptera. Journal of Applied Entomology 96:500–508
    [Google Scholar]
  27. Kronstad J. W., Schnepf H. E., Whiteley H. R. 1983; Diversity of locations for Bacillus thuringiensis crystal protein genes. Journal of Bacteriology 154:419–428
    [Google Scholar]
  28. Luthy P., Cordier J. L., Fischer H. M. 1982; Bacillus thuringiensis as a bacterial insecticide: basic consideration and applications. In Microbial and Viral Pesticides pp. 35–74 Kurstak E. Edited by New York: Marcel Dekker;
    [Google Scholar]
  29. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  30. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. L. 1987; Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. Journal of General Virology 68:1233–1250
    [Google Scholar]
  31. Miller L. K. 1988; Baculoviruses as gene expression vectors. Annual Review of Microbiology 42:177–199
    [Google Scholar]
  32. Newman G. R., Jasani B., Williams E. D. 1982; The preservation of ultrastructure and antigenicity. Journal of Microscopy 127:RP5–RP6
    [Google Scholar]
  33. Pennock G. D., Shoemaker C., Miller L. K. 1984; Strong and regulated expression of E. coli β-galactosidase in insect cells using a baculovirus vector. Molecular and Cellular Biology 4:399–406
    [Google Scholar]
  34. Possee R. D. 1986; Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Research 5:43–59
    [Google Scholar]
  35. Possee R. D., Howard S. C. 1987; Analysis of (he polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 15:10233–10248
    [Google Scholar]
  36. Possee R. D., Kelly D. C. 1988; Physical maps and comparative DNA hybridization of Mamestra brassicae and Panolis flammea nuclear polyhedrosis virus genomes. Journal of General Virology 69:1285–1298
    [Google Scholar]
  37. Schnepf H. E., Whiteley H. R. 1981; Cloning and expression of the Bacillus thuringiensis crystal protein gene. Journal of Biological Chemistry 260:6273–6280
    [Google Scholar]
  38. Smith G. E., Fraser M. J., Summers M. D. 1983a; Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: deletion mutations within the polyhedrin gene. Journal of Virology 46:584–593
    [Google Scholar]
  39. Smith G. E., Summers M. D., Fraser M. J. 1983b; Production of human β-interferon in insect cells with a baculovirus expression vector. Molecular and Cellular Biology 3:2156–2165
    [Google Scholar]
  40. Vaughn J. L., Goodwin R. H., Thompkins G. J., Mccawley P. 1977; The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13:213–217
    [Google Scholar]
  41. Vlak J. M., Klinkenberg F. A., Zaal K. J. M., Usmany M., Klinge-Roode E. C., Geervliet J. B. F., Roosien J., van Lent J. W. M. 1988; Functional studies on the plO gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-β-galactosidase fusion gene. Journal of General Virology 69:765–776
    [Google Scholar]
  42. Weyer U., Possee R. D. 1988; Functional analysis of the p10 gene 5′ leader sequence of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 16:661–666
    [Google Scholar]
  43. Weyer U., Possee R. D. 1989; Analysis of the promoter of the Autographa californica nuclear polyhedrosis virus p10 gene. Journal of General Virology 70:203–208
    [Google Scholar]
  44. Whiteley H. R., Kronstad J. W., Schnepf H. E., Desrosier J. P. 1982; Cloning the crystal protein gene of B. thuringiensis in E. coli . In Molecular Cloning and Gene Regulation in Bacilli pp. 131–144 Ganesan A. T., Chang S., Hoch J. A. Edited by New York & London: Academic Press;
    [Google Scholar]
  45. Wigley P. J. 1976 The epizootiology of a nuclear polyhedrosis virus disease of the winter moth Operophtera brumata L. at Wistman’s Wood, Dartmoor D. phil, thesis University of Oxford:
    [Google Scholar]
  46. Williams G. V., Rohel D. Z., Kuzio J., Faulkner P. 1989; A cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion/deletion mutants. Journal of General Virology 70:187–202
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-71-7-1535
Loading
/content/journal/jgv/10.1099/0022-1317-71-7-1535
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error