Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1993 Oct;67(10):5833–5842. doi: 10.1128/jvi.67.10.5833-5842.1993

Alternative transcriptional initiation as a novel mechanism for regulating expression of a baculovirus trans activator.

X Wu 1, S Stewart 1, D A Theilmann 1
PMCID: PMC238001  PMID: 8371344

Abstract

In this report, we show that the Orgyia pseudotsugata nuclear polyhedrosis virus p34 gene, which is homologous to the Autographa californica nuclear polyhedrosis virus PE-38 gene, is a trans activator. The predicted p34 protein contains a number of motifs that are similar to those found in other eukaryotic transcriptional trans activators, including a putative zinc finger DNA-binding domain, a glutamine-rich domain, and a leucine zipper. Northern (RNA) blot analysis showed that the p34 gene is expressed as a 1.1-kb mRNA from 1 to 48 h postinfection and as a 0.7-kb mRNA from 18 to 120 h postinfection. Mapping of these transcripts showed that they were 3' coterminal but initiated at different 5' start sites. The 1.1-kb transcript initiates at a baculovirus early gene motif (CACAGT) and encodes the entire p34 open reading frame (ORF). The 0.7-kb transcript initiates at a baculovirus late gene start site (GTAAG) internal to the p34 ORF. Western blot (immunoblot) analysis using p34 antisera showed that the 0.7-kb transcript is translated as an amino-terminally truncated 20-kDa form of the full length 34-kDa protein. Functional analysis indicated that the 34-kDa protein transcriptionally trans activates the IE-2 promoter whereas the 20-kDa protein does not. Therefore, p34 produces two functionally different proteins from the same ORF, using the novel mechanism of alternative transcriptional initiation.

Full text

PDF
5837

Images in this article

Selected References

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

  1. Bang D. D., Verhage R., Goosen N., Brouwer J., van de Putte P. Molecular cloning of RAD16, a gene involved in differential repair in Saccharomyces cerevisiae. Nucleic Acids Res. 1992 Aug 11;20(15):3925–3931. doi: 10.1093/nar/20.15.3925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blissard G. W., Quant-Russell R. L., Rohrmann G. F., Beaudreau G. S. Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata. Virology. 1989 Feb;168(2):354–362. doi: 10.1016/0042-6822(89)90276-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Blissard G. W., Rohrmann G. F. Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1989 Jun;170(2):537–555. doi: 10.1016/0042-6822(89)90445-5. [DOI] [PubMed] [Google Scholar]
  5. Carson D. D., Guarino L. A., Summers M. D. Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene. Virology. 1988 Feb;162(2):444–451. doi: 10.1016/0042-6822(88)90485-0. [DOI] [PubMed] [Google Scholar]
  6. Chisholm G. E., Henner D. J. Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene. J Virol. 1988 Sep;62(9):3193–3200. doi: 10.1128/jvi.62.9.3193-3200.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  8. Foulkes N. S., Sassone-Corsi P. More is better: activators and repressors from the same gene. Cell. 1992 Feb 7;68(3):411–414. doi: 10.1016/0092-8674(92)90178-f. [DOI] [PubMed] [Google Scholar]
  9. Freemont P. S., Hanson I. M., Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. doi: 10.1016/0092-8674(91)90229-r. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Fuchs L. Y., Woods M. S., Weaver R. F. Viral Transcription During Autographa californica Nuclear Polyhedrosis Virus Infection: a Novel RNA Polymerase Induced in Infected Spodoptera frugiperda Cells. J Virol. 1983 Dec;48(3):641–646. doi: 10.1128/jvi.48.3.641-646.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerster T., Balmaceda C. G., Roeder R. G. The cell type-specific octamer transcription factor OTF-2 has two domains required for the activation of transcription. EMBO J. 1990 May;9(5):1635–1643. doi: 10.1002/j.1460-2075.1990.tb08283.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goddard A. D., Borrow J., Freemont P. S., Solomon E. Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia. Science. 1991 Nov 29;254(5036):1371–1374. doi: 10.1126/science.1720570. [DOI] [PubMed] [Google Scholar]
  14. Grula M. A., Buller P. L., Weaver R. F. alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells. J Virol. 1981 Jun;38(3):916–921. doi: 10.1128/jvi.38.3.916-921.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guarino L. A., Summers M. D. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol. 1986 Feb;57(2):563–571. doi: 10.1128/jvi.57.2.563-571.1986. [DOI] [PMC free article] [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., Choi J., Guarino L. A., Summers M. D. Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein. J Virol. 1992 Dec;66(12):7429–7437. doi: 10.1128/jvi.66.12.7429-7437.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krappa R., Knebel-Mörsdorf D. Identification of the very early transcribed baculovirus gene PE-38. J Virol. 1991 Feb;65(2):805–812. doi: 10.1128/jvi.65.2.805-812.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Leisy D. J., Rohrmann G. F., Beaudreau G. S. Conservation of genome organization in two multicapsid nuclear polyhedrosis viruses. J Virol. 1984 Nov;52(2):699–702. doi: 10.1128/jvi.52.2.699-702.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin K. H., Cheng S. Y. An efficient method to purify active eukaryotic proteins from the inclusion bodies in Escherichia coli. Biotechniques. 1991 Dec;11(6):748, 750, 752-3. [PubMed] [Google Scholar]
  22. Lovering R., Hanson I. M., Borden K. L., Martin S., O'Reilly N. J., Evan G. I., Rahman D., Pappin D. J., Trowsdale J., Freemont P. S. Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2112–2116. doi: 10.1073/pnas.90.6.2112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lu A., Carstens E. B. Nucleotide sequence and transcriptional analysis of the p80 gene of Autographa californica nuclear polyhedrosis virus: a homologue of the Orgyia pseudotsugata nuclear polyhedrosis virus capsid-associated gene. Virology. 1992 Sep;190(1):201–209. doi: 10.1016/0042-6822(92)91206-a. [DOI] [PubMed] [Google Scholar]
  24. Lu A., Carstens E. B. Transcription analysis of the EcoRI D region of the baculovirus Autographa californica nuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene. J Virol. 1992 Feb;66(2):655–663. doi: 10.1128/jvi.66.2.655-663.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  26. O'Shea E. K., Rutkowski R., Kim P. S. Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. Cell. 1992 Feb 21;68(4):699–708. doi: 10.1016/0092-8674(92)90145-3. [DOI] [PubMed] [Google Scholar]
  27. Ooi B. G., Miller L. K. The influence of antisense RNA on transcriptional mapping of the 5' terminus of a baculovirus RNA. J Gen Virol. 1991 Mar;72(Pt 3):527–534. doi: 10.1099/0022-1317-72-3-527. [DOI] [PubMed] [Google Scholar]
  28. Passarelli A. L., Miller L. K. Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2. J Virol. 1993 Apr;67(4):2149–2158. doi: 10.1128/jvi.67.4.2149-2158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Quant-Russell R. L., Pearson M. N., Rohrmann G. F., Beaudreau G. S. Characterization of baculovirus p10 synthesis using monoclonal antibodies. Virology. 1987 Sep;160(1):9–19. doi: 10.1016/0042-6822(87)90038-9. [DOI] [PubMed] [Google Scholar]
  30. Ron D., Habener J. F. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 1992 Mar;6(3):439–453. doi: 10.1101/gad.6.3.439. [DOI] [PubMed] [Google Scholar]
  31. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  32. Theilmann D. A., Stewart S. Identification and characterization of the IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1991 Feb;180(2):492–508. doi: 10.1016/0042-6822(91)90063-h. [DOI] [PubMed] [Google Scholar]
  33. Theilmann D. A., Stewart S. Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1992 Mar;187(1):84–96. doi: 10.1016/0042-6822(92)90297-3. [DOI] [PubMed] [Google Scholar]
  34. Theilmann D. A., Stewart S. Tandemly repeated sequence at the 3' end of the IE-2 gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus is an enhancer element. Virology. 1992 Mar;187(1):97–106. doi: 10.1016/0042-6822(92)90298-4. [DOI] [PubMed] [Google Scholar]
  35. Thiem S. M., Miller L. K. A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper. J Virol. 1989 Nov;63(11):4489–4497. doi: 10.1128/jvi.63.11.4489-4497.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  37. Toneguzzo F., Glynn S., Levi E., Mjolsness S., Hayday A. Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA. Biotechniques. 1988 May;6(5):460–469. [PubMed] [Google Scholar]
  38. Xie W. Q., Rothblum L. I. Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques. 1991 Sep;11(3):324, 326-7. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES