Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Oct;67(10):5740–5748. doi: 10.1128/jvi.67.10.5740-5748.1993

Overexpression, purification, and late transcription factor activity of the 17-kilodalton protein encoded by the vaccinia virus A1L gene.

J G Keck 1, G R Kovacs 1, B Moss 1
PMCID: PMC237991  PMID: 8371339

Abstract

The A1L, A2L, and G8R open reading frames (ORFs) were previously shown by transfection assays to encode transactivators of late gene expression. We now present evidence that the 17-kDa protein product of the A1L gene can function in vitro as a transcription factor. Simultaneous overexpression of the transactivators was achieved by coinfecting HeLa cells with one recombinant vaccinia virus that encodes the bacteriophage T7 RNA polymerase and three recombinant vaccinia viruses that contain copies of A1L, A2L, and G8R ORFs regulated by T7 promoters. Extracts from the recombinant virus-infected cells exhibited greatly enhanced late in vitro transcription activity and served as a source of factors. The 17-kDa product of the A1L ORF represented approximately 8% of the ammonium sulfate-precipitated cell protein and copurified with a late transcription factor activity. The transcription factor activity could be specifically immunodepleted with immobilized antibody to the bacterially expressed A1L-encoded protein, providing additional evidence for its identity and role. A sequence encoding six consecutive histidines was added to the A1L ORF, which was then incorporated into the genome of a baculovirus expression vector. The 17-kDa protein, synthesized in insect cells and purified by binding to an Ni(2+)-chelating affinity column, could replace the vaccinia virus-overexpressed 17-kDa protein in transcription assays. In addition to the 17-kDa product of the A1L gene, which was named vaccinia virus late transcription factor 2, the proteins that stimulate specific transcription of late promoter-regulated templates included the viral multisubunit RNA polymerase, vaccinia virus late transcription factor 1 (the product of the G8R ORF), and at least one other partially purified protein.

Full text

PDF
5740

Images in this article

5743Image
on p.5743
5743Image
on p.5743
5744Image
on p.5744
5745Image
on p.5745
5746Image
on p.5746
5746Image
on p.5746

Selected References

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

  1. Ahn B. Y., Moss B. RNA polymerase-associated transcription specificity factor encoded by vaccinia virus. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3536–3540. doi: 10.1073/pnas.89.8.3536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldick C. J., Jr, Keck J. G., Moss B. Mutational analysis of the core, spacer, and initiator regions of vaccinia virus intermediate-class promoters. J Virol. 1992 Aug;66(8):4710–4719. doi: 10.1128/jvi.66.8.4710-4719.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldick C. J., Jr, Moss B. Characterization and temporal regulation of mRNAs encoded by vaccinia virus intermediate-stage genes. J Virol. 1993 Jun;67(6):3515–3527. doi: 10.1128/jvi.67.6.3515-3527.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broyles S. S., Fesler B. S. Vaccinia virus gene encoding a component of the viral early transcription factor. J Virol. 1990 Apr;64(4):1523–1529. doi: 10.1128/jvi.64.4.1523-1529.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broyles S. S., Li J., Moss B. Promoter DNA contacts made by the vaccinia virus early transcription factor. J Biol Chem. 1991 Aug 15;266(23):15539–15544. [PubMed] [Google Scholar]
  6. Broyles S. S., Moss B. DNA-dependent ATPase activity associated with vaccinia virus early transcription factor. J Biol Chem. 1988 Aug 5;263(22):10761–10765. [PubMed] [Google Scholar]
  7. Carpenter M. S., DeLange A. M. Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression. Virology. 1992 May;188(1):233–244. doi: 10.1016/0042-6822(92)90753-c. [DOI] [PubMed] [Google Scholar]
  8. Cochran M. A., Mackett M., Moss B. Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. Proc Natl Acad Sci U S A. 1985 Jan;82(1):19–23. doi: 10.1073/pnas.82.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davison A. J., Moss B. Structure of vaccinia virus early promoters. J Mol Biol. 1989 Dec 20;210(4):749–769. doi: 10.1016/0022-2836(89)90107-1. [DOI] [PubMed] [Google Scholar]
  10. Davison A. J., Moss B. Structure of vaccinia virus late promoters. J Mol Biol. 1989 Dec 20;210(4):771–784. doi: 10.1016/0022-2836(89)90108-3. [DOI] [PubMed] [Google Scholar]
  11. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  12. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gershon P. D., Moss B. Early transcription factor subunits are encoded by vaccinia virus late genes. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4401–4405. doi: 10.1073/pnas.87.11.4401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harris N., Rosales R., Moss B. Transcription initiation factor activity of vaccinia virus capping enzyme is independent of mRNA guanylylation. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2860–2864. doi: 10.1073/pnas.90.7.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hochuli E., Döbeli H., Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 1987 Dec 18;411:177–184. doi: 10.1016/s0021-9673(00)93969-4. [DOI] [PubMed] [Google Scholar]
  17. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Janknecht R., de Martynoff G., Lou J., Hipskind R. A., Nordheim A., Stunnenberg H. G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972–8976. doi: 10.1073/pnas.88.20.8972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Keck J. G., Baldick C. J., Jr, Moss B. Role of DNA replication in vaccinia virus gene expression: a naked template is required for transcription of three late trans-activator genes. Cell. 1990 Jun 1;61(5):801–809. doi: 10.1016/0092-8674(90)90190-p. [DOI] [PubMed] [Google Scholar]
  20. Keck J. G., Feigenbaum F., Moss B. Mutational analysis of a predicted zinc-binding motif in the 26-kilodalton protein encoded by the vaccinia virus A2L gene: correlation of zinc binding with late transcriptional transactivation activity. J Virol. 1993 Oct;67(10):5749–5753. doi: 10.1128/jvi.67.10.5749-5753.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  23. Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moss B., Ahn B. Y., Amegadzie B., Gershon P. D., Keck J. G. Cytoplasmic transcription system encoded by vaccinia virus. J Biol Chem. 1991 Jan 25;266(3):1355–1358. [PubMed] [Google Scholar]
  25. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
  26. Moss B. Regulation of vaccinia virus transcription. Annu Rev Biochem. 1990;59:661–688. doi: 10.1146/annurev.bi.59.070190.003305. [DOI] [PubMed] [Google Scholar]
  27. Shuman S., Moss B. Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. J Biol Chem. 1988 May 5;263(13):6220–6225. [PubMed] [Google Scholar]
  28. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  29. Vos J. C., Sasker M., Stunnenberg H. G. Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase. Cell. 1991 Apr 5;65(1):105–113. doi: 10.1016/0092-8674(91)90412-r. [DOI] [PubMed] [Google Scholar]
  30. Vos J. C., Stunnenberg H. G. Derepression of a novel class of vaccinia virus genes upon DNA replication. EMBO J. 1988 Nov;7(11):3487–3492. doi: 10.1002/j.1460-2075.1988.tb03224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wright C. F., Keck J. G., Tsai M. M., Moss B. A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene. J Virol. 1991 Jul;65(7):3715–3720. doi: 10.1128/jvi.65.7.3715-3720.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wright C. F., Moss B. Identification of factors specific for transcription of the late class of vaccinia virus genes. J Virol. 1989 Oct;63(10):4224–4233. doi: 10.1128/jvi.63.10.4224-4233.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhang Y., Keck J. G., Moss B. Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus. J Virol. 1992 Nov;66(11):6470–6479. doi: 10.1128/jvi.66.11.6470-6479.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES