Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1996 Nov;70(11):7669–7677. doi: 10.1128/jvi.70.11.7669-7677.1996

De novo synthesis of the early transcription factor 70-kilodalton subunit is required for morphogenesis of vaccinia virions.

X Hu 1, L J Carroll 1, E J Wolffe 1, B Moss 1
PMCID: PMC190836  PMID: 8892887

Abstract

Vaccinia virus early transcription factor (VETF) is a heterodimeric protein that is packaged in virus particles for expression of early genes during the next round of infection. To investigate additional roles of VETF, we constructed a conditionally lethal recombinant vaccinia virus in which the D6R gene, encoding the 70-kDa subunit of VETF, is under stringent Escherichia coli lac operator control. When cells were infected with the recombinant virus in the absence of an inducer, synthesis of the 70-kDa protein was undetectable and the yield of infectious virus was severely reduced. Under these nonpermissive conditions, DNA replication and synthesis of viral proteins other than the one encoded by D6R occurred, suggesting that de novo synthesis of VETF is not required for expression of early or late genes during the virus growth cycle. Electron microscopy, however, revealed that immature virus particles and masses of electron-dense material accumulated in the absence of an inducer. We concluded that VETF has a direct role in virion morphogenesis or is required for expression of a novel subset of genes that have such a role.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Ahn B. Y., Gershon P. D., Moss B. RNA polymerase-associated protein Rap94 confers promoter specificity for initiating transcription of vaccinia virus early stage genes. J Biol Chem. 1994 Mar 11;269(10):7552–7557. [PubMed] [Google Scholar]
  2. 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]
  3. Alexander W. A., Moss B., Fuerst T. R. Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor. J Virol. 1992 May;66(5):2934–2942. doi: 10.1128/jvi.66.5.2934-2942.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baldick C. J., Jr, Cassetti M. C., Harris N., Moss B. Ordered assembly of a functional preinitiation transcription complex, containing vaccinia virus early transcription factor and RNA polymerase, on an immobilized template. J Virol. 1994 Sep;68(9):6052–6056. doi: 10.1128/jvi.68.9.6052-6056.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barbosa E., Moss B. mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Purification and physical properties. J Biol Chem. 1978 Nov 10;253(21):7692–7697. [PubMed] [Google Scholar]
  6. Baroudy B. M., Moss B. Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions. J Biol Chem. 1980 May 10;255(9):4372–4380. [PubMed] [Google Scholar]
  7. Broyles S. S. A role for ATP hydrolysis in vaccinia virus early gene transcription. Dissociation of the early transcription factor-promoter complex. J Biol Chem. 1991 Aug 15;266(23):15545–15548. [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Broyles S. S., Yuen L., Shuman S., Moss B. Purification of a factor required for transcription of vaccinia virus early genes. J Biol Chem. 1988 Aug 5;263(22):10754–10760. [PubMed] [Google Scholar]
  11. Deng L., Shuman S. A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter. J Biol Chem. 1994 May 13;269(19):14323–14328. [PubMed] [Google Scholar]
  12. 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]
  13. Franke C. A., Rice C. M., Strauss J. H., Hruby D. E. Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. Mol Cell Biol. 1985 Aug;5(8):1918–1924. doi: 10.1128/mcb.5.8.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuerst T. R., Fernandez M. P., Moss B. Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2549–2553. doi: 10.1073/pnas.86.8.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garcés J., Masternak K., Kunz B., Wittek R. Reactivation of transcription from a vaccinia virus early promoter late in infection. J Virol. 1993 Sep;67(9):5394–5401. doi: 10.1128/jvi.67.9.5394-5401.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gershon P. D., Ahn B. Y., Garfield M., Moss B. Poly(A) polymerase and a dissociable polyadenylation stimulatory factor encoded by vaccinia virus. Cell. 1991 Sep 20;66(6):1269–1278. doi: 10.1016/0092-8674(91)90048-4. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Grimley P. M., Rosenblum E. N., Mims S. J., Moss B. Interruption by Rifampin of an early stage in vaccinia virus morphogenesis: accumulation of membranes which are precursors of virus envelopes. J Virol. 1970 Oct;6(4):519–533. doi: 10.1128/jvi.6.4.519-533.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kates J. R., McAuslan B. R. Poxvirus DNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1967 Jul;58(1):134–141. doi: 10.1073/pnas.58.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Keck J. G., Kovacs G. R., Moss B. Overexpression, purification, and late transcription factor activity of the 17-kilodalton protein encoded by the vaccinia virus A1L gene. J Virol. 1993 Oct;67(10):5740–5748. doi: 10.1128/jvi.67.10.5740-5748.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovacs G. R., Rosales R., Keck J. G., Moss B. Modification of the cascade model for regulation of vaccinia virus gene expression: purification of a prereplicative, late-stage-specific transcription factor. J Virol. 1994 May;68(5):3443–3447. doi: 10.1128/jvi.68.5.3443-3447.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li J., Broyles S. S. Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor. J Biol Chem. 1993 Feb 5;268(4):2773–2780. [PubMed] [Google Scholar]
  24. Li J., Broyles S. S. The DNA-dependent ATPase activity of vaccinia virus early gene transcription factor is essential for its transcription activation function. J Biol Chem. 1993 Sep 25;268(27):20016–20021. [PubMed] [Google Scholar]
  25. Li J., Pennington M. J., Broyles S. S. Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components. J Virol. 1994 Apr;68(4):2605–2614. doi: 10.1128/jvi.68.4.2605-2614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martin S. A., Paoletti E., Moss B. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9322–9329. [PubMed] [Google Scholar]
  27. Morgan C. The insertion of DNA into vaccinia virus. Science. 1976 Aug 13;193(4253):591–592. doi: 10.1126/science.959819. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Moss B., Rosenblum E. N., Paoletti E. Polyadenylate polymerase from vaccinia virions. Nat New Biol. 1973 Sep 12;245(141):59–63. doi: 10.1038/newbio245059a0. [DOI] [PubMed] [Google Scholar]
  30. Munyon W., Paoletti E., Grace J. T., Jr RNA polymerase activity in purified infectious vaccinia virus. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2280–2287. doi: 10.1073/pnas.58.6.2280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rodriguez J. F., Smith G. L. IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress. Nucleic Acids Res. 1990 Sep 25;18(18):5347–5351. doi: 10.1093/nar/18.18.5347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rosales R., Harris N., Ahn B. Y., Moss B. Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit. J Biol Chem. 1994 May 13;269(19):14260–14267. [PubMed] [Google Scholar]
  34. Rosales R., Sutter G., Moss B. A cellular factor is required for transcription of vaccinia viral intermediate-stage genes. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3794–3798. doi: 10.1073/pnas.91.9.3794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schnierle B. S., Gershon P. D., Moss B. Cap-specific mRNA (nucleoside-O2'-)-methyltransferase and poly(A) polymerase stimulatory activities of vaccinia virus are mediated by a single protein. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2897–2901. doi: 10.1073/pnas.89.7.2897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shuman S., Broyles S. S., Moss B. Purification and characterization of a transcription termination factor from vaccinia virions. J Biol Chem. 1987 Sep 5;262(25):12372–12380. [PubMed] [Google Scholar]
  37. Spencer E., Shuman S., Hurwitz J. Purification and properties of vaccinia virus DNA-dependent RNA polymerase. J Biol Chem. 1980 Jun 10;255(11):5388–5395. [PubMed] [Google Scholar]
  38. 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]
  39. Vos J. C., Sasker M., Stunnenberg H. G. Vaccinia virus capping enzyme is a transcription initiation factor. EMBO J. 1991 Sep;10(9):2553–2558. doi: 10.1002/j.1460-2075.1991.tb07795.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ward G. A., Stover C. K., Moss B., Fuerst T. R. Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6773–6777. doi: 10.1073/pnas.92.15.6773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wolffe E. J., Moore D. M., Peters P. J., Moss B. Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis. J Virol. 1996 May;70(5):2797–2808. doi: 10.1128/jvi.70.5.2797-2808.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wright C. F., Coroneos A. M. Purification of the late transcription system of vaccinia virus: identification of a novel transcription factor. J Virol. 1993 Dec;67(12):7264–7270. doi: 10.1128/jvi.67.12.7264-7270.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Yuen L., Davison A. J., Moss B. Early promoter-binding factor from vaccinia virions. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6069–6073. doi: 10.1073/pnas.84.17.6069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang Y. F., Moss B. Inducer-dependent conditional-lethal mutant animal viruses. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1511–1515. doi: 10.1073/pnas.88.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang Y., Ahn B. Y., Moss B. Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein. J Virol. 1994 Mar;68(3):1360–1370. doi: 10.1128/jvi.68.3.1360-1370.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES