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. 1988 Jan;62(1):297–304. doi: 10.1128/jvi.62.1.297-304.1988

Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster.

J N Miner 1, S L Weinrich 1, D E Hruby 1
PMCID: PMC250530  PMID: 3334746

Abstract

Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.

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Selected References

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

  1. Alwine J. C., Picardi J. Activity of simian virus 40 late promoter elements in the absence of large T antigen: evidence for repression of late gene expression. J Virol. 1986 Nov;60(2):400–404. doi: 10.1128/jvi.60.2.400-404.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertholet C., Drillien R., Wittek R. One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2096–2100. doi: 10.1073/pnas.82.7.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertholet C., Stocco P., Van Meir E., Wittek R. Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. EMBO J. 1986 Aug;5(8):1951–1957. doi: 10.1002/j.1460-2075.1986.tb04449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bertholet C., Van Meir E., ten Heggeler-Bordier B., Wittek R. Vaccinia virus produces late mRNAs by discontinuous synthesis. Cell. 1987 Jul 17;50(2):153–162. doi: 10.1016/0092-8674(87)90211-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chipchase M., Schwendimann F., Wyler R. A map of the late proteins of vaccinia virus. Virology. 1980 Aug;105(1):261–264. doi: 10.1016/0042-6822(80)90176-2. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Coen D. M., Weinheimer S. P., McKnight S. L. A genetic approach to promoter recognition during trans induction of viral gene expression. Science. 1986 Oct 3;234(4772):53–59. doi: 10.1126/science.3018926. [DOI] [PubMed] [Google Scholar]
  9. Cooper J. A., Wittek R., Moss B. Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs. J Virol. 1981 Sep;39(3):733–745. doi: 10.1128/jvi.39.3.733-745.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hodges W. M., Hruby D. E. Cell-free translation of a chimeric eucaryotic-procaryotic message yields functional chloramphenicol acetyltransferase. Anal Biochem. 1987 Jan;160(1):65–67. doi: 10.1016/0003-2697(87)90614-2. [DOI] [PubMed] [Google Scholar]
  14. Hruby D. E., Guarino L. A., Kates J. R. Vaccinia virus replication. I. Requirement for the host-cell nucleus. J Virol. 1979 Feb;29(2):705–715. doi: 10.1128/jvi.29.2.705-715.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hruby D. E., Lynn D. L., Kates J. R. Vaccinia virus replication requires active participation of the host cell transcriptional apparatus. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1887–1890. doi: 10.1073/pnas.76.4.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hänggi M., Bannwarth W., Stunnenberg H. G. Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J. 1986 May;5(5):1071–1076. doi: 10.1002/j.1460-2075.1986.tb04324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Isle H. B., Venkatesan S., Moss B. Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome. Virology. 1981 Jul 15;112(1):306–317. doi: 10.1016/0042-6822(81)90636-x. [DOI] [PubMed] [Google Scholar]
  18. Keller J. M., Alwine J. C. Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation. Mol Cell Biol. 1985 Aug;5(8):1859–1869. doi: 10.1128/mcb.5.8.1859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Mahr A., Roberts B. E. Arrangement of late RNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment. J Virol. 1984 Feb;49(2):510–520. doi: 10.1128/jvi.49.2.510-520.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mahr A., Roberts B. E. Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment. J Virol. 1984 Feb;49(2):497–509. doi: 10.1128/jvi.49.2.497-509.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  23. Moss B., Salzman N. P. Sequential protein synthesis following vaccinia virus infection. J Virol. 1968 Oct;2(10):1016–1027. doi: 10.1128/jvi.2.10.1016-1027.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oda K. I., Joklik W. K. Hybridization and sedimentation studies on "early" and "late" vaccinia messenger RNA. J Mol Biol. 1967 Aug 14;27(3):395–419. doi: 10.1016/0022-2836(67)90047-2. [DOI] [PubMed] [Google Scholar]
  25. Paoletti E., Grady L. J. Transcriptional complexity of vaccinia virus in vivo and in vitro. J Virol. 1977 Sep;23(3):608–615. doi: 10.1128/jvi.23.3.608-615.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pennington T. H., Follett E. A. Vaccinia virus replication in enucleate BSC-1 cells: particle production and synthesis of viral DNA and proteins. J Virol. 1974 Feb;13(2):488–493. doi: 10.1128/jvi.13.2.488-493.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosel J. L., Earl P. L., Weir J. P., Moss B. Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. J Virol. 1986 Nov;60(2):436–449. doi: 10.1128/jvi.60.2.436-449.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Salzman N. P., Sebring E. D. Sequential formation of vaccinia virus proteins and viral deoxyribonucleic acid replication. J Virol. 1967 Feb;1(1):16–23. doi: 10.1128/jvi.1.1.16-23.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwer B., Visca P., Vos J. C., Stunnenberg H. G. Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader. Cell. 1987 Jul 17;50(2):163–169. doi: 10.1016/0092-8674(87)90212-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Silver M., Dales S. Evidence against involvement of host transcription in the replication of vaccinia and herpes simplex viruses. Virology. 1982 Apr 15;118(1):214–218. doi: 10.1016/0042-6822(82)90334-8. [DOI] [PubMed] [Google Scholar]
  31. Silver M., McFadden G., Wilton S., Dales S. Biogenesis of poxviruses: role for the DNA-dependent RNA polymerase II of the host during expression of late functions. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4122–4125. doi: 10.1073/pnas.76.8.4122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tartaglia J., Paoletti E. Physical mapping and DNA sequence analysis of the rifampicin resistance locus in vaccinia virus. Virology. 1985 Dec;147(2):394–404. doi: 10.1016/0042-6822(85)90141-2. [DOI] [PubMed] [Google Scholar]
  33. Tartaglia J., Piccini A., Paoletti E. Vaccinia virus rifampicin-resistance locus specifies a late 63,000 Da gene product. Virology. 1986 Apr 15;150(1):45–54. doi: 10.1016/0042-6822(86)90264-3. [DOI] [PubMed] [Google Scholar]
  34. Weinrich S. L., Hruby D. E. Noncoordinate regulation of a vaccinia virus late gene cluster. J Virol. 1987 Mar;61(3):639–645. doi: 10.1128/jvi.61.3.639-645.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weinrich S. L., Niles E. G., Hruby D. E. Transcriptional and translational analysis of the vaccinia virus late gene L65. J Virol. 1985 Aug;55(2):450–457. doi: 10.1128/jvi.55.2.450-457.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weir J. P., Moss B. Determination of the transcriptional regulatory region of a vaccinia virus late gene. J Virol. 1987 Jan;61(1):75–80. doi: 10.1128/jvi.61.1.75-80.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wertz G. W., Stott E. J., Young K. K., Anderson K., Ball L. A. Expression of the fusion protein of human respiratory syncytial virus from recombinant vaccinia virus vectors and protection of vaccinated mice. J Virol. 1987 Feb;61(2):293–301. doi: 10.1128/jvi.61.2.293-301.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wittek R. Organization and expression of the poxvirus genome. Experientia. 1982 Mar 15;38(3):285–297. doi: 10.1007/BF01949349. [DOI] [PubMed] [Google Scholar]

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