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. 1983 May;46(2):584–593. doi: 10.1128/jvi.46.2.584-593.1983

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

Gale E Smith 1, M J Fraser 1, Max D Summers 1
PMCID: PMC255161  PMID: 16789242

Abstract

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.

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Selected References

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

  1. Adang M. J., Miller L. K. Molecular cloning of DNA complementary to mRNA of the baculovirus Autographa californica nuclear polyhedrosis virus: location and gene products of RNA transcripts found late in infection. J Virol. 1982 Dec;44(3):782–793. doi: 10.1128/jvi.44.3.782-793.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown M., Crawford A. M., Faulkner P. Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups. J Virol. 1979 Jul;31(1):190–198. doi: 10.1128/jvi.31.1.190-198.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carstens E. B. Mapping the Mutation Site of an Autographa californica Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant. J Virol. 1982 Sep;43(3):809–818. doi: 10.1128/jvi.43.3.809-818.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carstens E. B., Tjia S. T., Doerfler W. Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus I. Synthesis of intracellular proteins after virus infection. Virology. 1979 Dec;99(2):386–398. doi: 10.1016/0042-6822(79)90017-5. [DOI] [PubMed] [Google Scholar]
  5. Cochran M. A., Carstens E. B., Eaton B. T., Faulkner P. Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA. J Virol. 1982 Mar;41(3):940–946. doi: 10.1128/jvi.41.3.940-946.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S. N., Chang A. C., Boyer H. W., Helling R. B. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240–3244. doi: 10.1073/pnas.70.11.3240. [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. Hink W. F. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature. 1970 May 2;226(5244):466–467. doi: 10.1038/226466b0. [DOI] [PubMed] [Google Scholar]
  9. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lee H. H., Miller L. K. Isolation, Complementation, and Initial Characterization of Temperature-Sensitive Mutants of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus. J Virol. 1979 Jul;31(1):240–252. doi: 10.1128/jvi.31.1.240-252.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. MOLLENHAUER H. H. PLASTIC EMBEDDING MIXTURES FOR USE IN ELECTRON MICROSCOPY. Stain Technol. 1964 Mar;39:111–114. [PubMed] [Google Scholar]
  13. McKnight S. L., Gavis E. R. Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro. Nucleic Acids Res. 1980 Dec 20;8(24):5931–5948. doi: 10.1093/nar/8.24.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miller D. W., Miller L. K. A virus mutant with an insertion of a copia-like transposable element. Nature. 1982 Oct 7;299(5883):562–564. doi: 10.1038/299562a0. [DOI] [PubMed] [Google Scholar]
  15. Mocarski E. S., Post L. E., Roizman B. Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions. Cell. 1980 Nov;22(1 Pt 1):243–255. doi: 10.1016/0092-8674(80)90172-5. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Potter K. N., Faulkner P., MacKinnon E. A. Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus. J Virol. 1976 Jun;18(3):1040–1050. doi: 10.1128/jvi.18.3.1040-1050.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith G. E., Summers M. D. Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants. J Virol. 1981 Jul;39(1):125–137. doi: 10.1128/jvi.39.1.125-137.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smith G. E., Summers M. D. Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. J Virol. 1979 Jun;30(3):828–838. doi: 10.1128/jvi.30.3.828-838.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smith G. E., Vlak J. M., Summers M. D. In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs. J Virol. 1982 Oct;44(1):199–208. doi: 10.1128/jvi.44.1.199-208.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith G. E., Vlak J. M., Summers M. D. Physical Analysis of Autographa californica Nuclear Polyhedrosis Virus Transcripts for Polyhedrin and 10,000-Molecular-Weight Protein. J Virol. 1983 Jan;45(1):215–225. doi: 10.1128/jvi.45.1.215-225.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Summers M. D., Volkman L. E., Hsieh C. Immunoperoxidase detection of baculovirus antigens in insect cells. J Gen Virol. 1978 Sep;40(3):545–557. doi: 10.1099/0022-1317-40-3-545. [DOI] [PubMed] [Google Scholar]
  23. Vlak J. M., Smith G. E. Orientation of the Genome of Autographa californica Nuclear Polyhedrosis Virus: a Proposal. J Virol. 1982 Mar;41(3):1118–1121. doi: 10.1128/jvi.41.3.1118-1121.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vlak J. M., Smith G. E., Summers M. D. Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA. J Virol. 1981 Dec;40(3):762–771. doi: 10.1128/jvi.40.3.762-771.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Volkman L. E., Summers M. D., Hsieh C. H. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies. J Virol. 1976 Sep;19(3):820–832. doi: 10.1128/jvi.19.3.820-832.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Volkman L. E., Summers M. D., Hsieh C. H. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies. J Virol. 1976 Sep;19(3):820–832. doi: 10.1128/jvi.19.3.820-832.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weir J. P., Bajszár G., Moss B. Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1210–1214. doi: 10.1073/pnas.79.4.1210. [DOI] [PMC free article] [PubMed] [Google Scholar]

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