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Journal of Virology logoLink to Journal of Virology
. 1992 Sep;66(9):5525–5533. doi: 10.1128/jvi.66.9.5525-5533.1992

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

P A Hershberger 1, J A Dickson 1, P D Friesen 1
PMCID: PMC289111  PMID: 1501287

Abstract

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.

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Selected References

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  1. Blissard G. W., Rohrmann G. F. Baculovirus diversity and molecular biology. Annu Rev Entomol. 1990;35:127–155. doi: 10.1146/annurev.en.35.010190.001015. [DOI] [PubMed] [Google Scholar]
  2. Carson D. D., Summers M. D., Guarino L. A. Molecular analysis of a baculovirus regulatory gene. Virology. 1991 May;182(1):279–286. doi: 10.1016/0042-6822(91)90671-w. [DOI] [PubMed] [Google Scholar]
  3. Clem R. J., Fechheimer M., Miller L. K. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science. 1991 Nov 29;254(5036):1388–1390. doi: 10.1126/science.1962198. [DOI] [PubMed] [Google Scholar]
  4. Crawford A. M., Miller L. K. Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1988 Aug;62(8):2773–2781. doi: 10.1128/jvi.62.8.2773-2781.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dickson J. A., Friesen P. D. Identification of upstream promoter elements mediating early transcription from the 35,000-molecular-weight protein gene of Autographa californica nuclear polyhedrosis virus. J Virol. 1991 Aug;65(8):4006–4016. doi: 10.1128/jvi.65.8.4006-4016.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Friesen P. D., Miller L. K. Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindIII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1987 Jul;61(7):2264–2272. doi: 10.1128/jvi.61.7.2264-2272.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friesen P. D., Miller L. K. The regulation of baculovirus gene expression. Curr Top Microbiol Immunol. 1986;131:31–49. doi: 10.1007/978-3-642-71589-1_3. [DOI] [PubMed] [Google Scholar]
  8. Friesen P. D., Nissen M. S. Gene organization and transcription of TED, a lepidopteran retrotransposon integrated within the baculovirus genome. Mol Cell Biol. 1990 Jun;10(6):3067–3077. doi: 10.1128/mcb.10.6.3067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friesen P. D., Rice W. C., Miller D. W., Miller L. K. Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites. Mol Cell Biol. 1986 May;6(5):1599–1607. doi: 10.1128/mcb.6.5.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gordon J. D., Carstens E. B. Phenotypic characterization and physical mapping of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus defective in DNA synthesis. Virology. 1984 Oct 15;138(1):69–81. doi: 10.1016/0042-6822(84)90148-x. [DOI] [PubMed] [Google Scholar]
  11. Gregory C. D., Dive C., Henderson S., Smith C. A., Williams G. T., Gordon J., Rickinson A. B. Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature. 1991 Feb 14;349(6310):612–614. doi: 10.1038/349612a0. [DOI] [PubMed] [Google Scholar]
  12. Guarino L. A., Summers M. D. Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol. 1987 Jul;61(7):2091–2099. doi: 10.1128/jvi.61.7.2091-2099.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Henderson S., Rowe M., Gregory C., Croom-Carter D., Wang F., Longnecker R., Kieff E., Rickinson A. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell. 1991 Jun 28;65(7):1107–1115. doi: 10.1016/0092-8674(91)90007-l. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  16. Huh N. E., Weaver R. F. Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus. J Gen Virol. 1990 Jan;71(Pt 1):195–201. doi: 10.1099/0022-1317-71-1-195. [DOI] [PubMed] [Google Scholar]
  17. Kovacs G. R., Guarino L. A., Summers M. D. Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus. J Virol. 1991 Oct;65(10):5281–5288. doi: 10.1128/jvi.65.10.5281-5288.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee H. H., Miller L. K. Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J Virol. 1978 Sep;27(3):754–767. doi: 10.1128/jvi.27.3.754-767.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lu A., Carstens E. B. Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus. Virology. 1991 Mar;181(1):336–347. doi: 10.1016/0042-6822(91)90500-b. [DOI] [PubMed] [Google Scholar]
  20. Martz E., Howell D. M. CTL: virus control cells first and cytolytic cells second? DNA fragmentation, apoptosis and the prelytic halt hypothesis. Immunol Today. 1989 Mar;10(3):79–86. doi: 10.1016/0167-5699(89)90231-4. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Nissen M. S., Friesen P. D. Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. J Virol. 1989 Feb;63(2):493–503. doi: 10.1128/jvi.63.2.493-503.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nuñez G., Hockenbery D., McDonnell T. J., Sorensen C. M., Korsmeyer S. J. Bcl-2 maintains B cell memory. Nature. 1991 Sep 5;353(6339):71–73. doi: 10.1038/353071a0. [DOI] [PubMed] [Google Scholar]
  24. O'Reilly D. R., Miller L. K. A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase. Science. 1989 Sep 8;245(4922):1110–1112. doi: 10.1126/science.2505387. [DOI] [PubMed] [Google Scholar]
  25. Pennock G. D., Shoemaker C., Miller L. K. Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol Cell Biol. 1984 Mar;4(3):399–406. doi: 10.1128/mcb.4.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rice W. C., Miller L. K. Baculovirus transcription in the presence of inhibitors and in nonpermissive Drosophila cells. Virus Res. 1986 Nov;6(2):155–172. doi: 10.1016/0168-1702(86)90047-x. [DOI] [PubMed] [Google Scholar]
  27. Sellins K. S., Cohen J. J. Polyomavirus DNA is damaged in target cells during cytotoxic T-lymphocyte-mediated killing. J Virol. 1989 Feb;63(2):572–578. doi: 10.1128/jvi.63.2.572-578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  29. Tomalski M. D., Wu J. G., Miller L. K. The location, sequence, transcription, and regulation of a baculovirus DNA polymerase gene. Virology. 1988 Dec;167(2):591–600. [PubMed] [Google Scholar]
  30. Vaughn J. L., Goodwin R. H., Tompkins G. J., McCawley P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro. 1977 Apr;13(4):213–217. doi: 10.1007/BF02615077. [DOI] [PubMed] [Google Scholar]
  31. White E., Sabbatini P., Debbas M., Wold W. S., Kusher D. I., Gooding L. R. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol Cell Biol. 1992 Jun;12(6):2570–2580. doi: 10.1128/mcb.12.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wold W. S., Gooding L. R. Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology. 1991 Sep;184(1):1–8. doi: 10.1016/0042-6822(91)90815-s. [DOI] [PubMed] [Google Scholar]

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