Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Jun;71(6):4300–4309. doi: 10.1128/jvi.71.6.4300-4309.1997

The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation.

D J Pereira 1, N Muzyczka 1
PMCID: PMC191646  PMID: 9151818

Abstract

We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Beaton A., Palumbo P., Berns K. I. Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein. J Virol. 1989 Oct;63(10):4450–4454. doi: 10.1128/jvi.63.10.4450-4454.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becerra S. P., Koczot F., Fabisch P., Rose J. A. Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. J Virol. 1988 Aug;62(8):2745–2754. doi: 10.1128/jvi.62.8.2745-2754.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Becerra S. P., Rose J. A., Hardy M., Baroudy B. M., Anderson C. W. Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7919–7923. doi: 10.1073/pnas.82.23.7919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolwig G. M., Bruder J. T., Hearing P. Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A. Nucleic Acids Res. 1992 Dec 25;20(24):6555–6564. doi: 10.1093/nar/20.24.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  6. Bruder J. T., Hearing P. Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions. Mol Cell Biol. 1989 Nov;9(11):5143–5153. doi: 10.1128/mcb.9.11.5143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter B. J., Antoni B. A., Klessig D. F. Adenovirus containing a deletion of the early region 2A gene allows growth of adeno-associated virus with decreased efficiency. Virology. 1992 Nov;191(1):473–476. doi: 10.1016/0042-6822(92)90213-9. [DOI] [PubMed] [Google Scholar]
  8. Carter B. J., Marcus-Sekura C. J., Laughlin C. A., Ketner G. Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication. Virology. 1983 Apr 30;126(2):505–516. doi: 10.1016/s0042-6822(83)80008-7. [DOI] [PubMed] [Google Scholar]
  9. Casto B. C., Atchison R. W., Hammon W. M. Studies on the relationship between adeno-associated virus type I (AAV-1) and adenoviruses. I. Replication of AAV-1 in certain cell cultures and its effect on helper adenovirus. Virology. 1967 May;32(1):52–59. doi: 10.1016/0042-6822(67)90251-6. [DOI] [PubMed] [Google Scholar]
  10. Chang L. S., Shenk T. The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J Virol. 1990 May;64(5):2103–2109. doi: 10.1128/jvi.64.5.2103-2109.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang L. S., Shi Y., Shenk T. Adeno-associated virus P5 promoter contains an adenovirus E1A-inducible element and a binding site for the major late transcription factor. J Virol. 1989 Aug;63(8):3479–3488. doi: 10.1128/jvi.63.8.3479-3488.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chodosh L. A., Carthew R. W., Sharp P. A. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol Cell Biol. 1986 Dec;6(12):4723–4733. doi: 10.1128/mcb.6.12.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  14. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao X., Huang L. A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun. 1991 Aug 30;179(1):280–285. doi: 10.1016/0006-291x(91)91366-k. [DOI] [PubMed] [Google Scholar]
  16. Gavin B. J., Ward D. C. Positive and negative regulation of the minute virus of mice P38 promoter. J Virol. 1990 May;64(5):2057–2063. doi: 10.1128/jvi.64.5.2057-2063.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gu M. L., Chen F. X., Rhode S. L. Parvovirus H-1 P38 promoter requires the trans-activation region (tar), an SP1 site, and a TATA box for full activity. Virology. 1992 Mar;187(1):10–17. doi: 10.1016/0042-6822(92)90290-6. [DOI] [PubMed] [Google Scholar]
  18. Hagen G., Müller S., Beato M., Suske G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 1992 Nov 11;20(21):5519–5525. doi: 10.1093/nar/20.21.5519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hermonat P. L., Labow M. A., Wright R., Berns K. I., Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 1984 Aug;51(2):329–339. doi: 10.1128/jvi.51.2.329-339.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hermonat P. L., Santin A. D., Batchu R. B. The adeno-associated virus Rep78 major regulatory/transformation suppressor protein binds cellular Sp1 in vitro and evidence of a biological effect. Cancer Res. 1996 Nov 15;56(22):5299–5304. [PubMed] [Google Scholar]
  21. Hörer M., Weger S., Butz K., Hoppe-Seyler F., Geisen C., Kleinschmidt J. A. Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters. J Virol. 1995 Sep;69(9):5485–5496. doi: 10.1128/jvi.69.9.5485-5496.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Im D. S., Muzyczka N. Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization. J Virol. 1992 Feb;66(2):1119–1128. doi: 10.1128/jvi.66.2.1119-1128.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Janik J. E., Huston M. M., Cho K., Rose J. A. Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA. Virology. 1989 Feb;168(2):320–329. doi: 10.1016/0042-6822(89)90272-9. [DOI] [PubMed] [Google Scholar]
  24. Janik J. E., Huston M. M., Rose J. A. Adeno-associated virus proteins: origin of the capsid components. J Virol. 1984 Nov;52(2):591–597. doi: 10.1128/jvi.52.2.591-597.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kingsley C., Winoto A. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol. 1992 Oct;12(10):4251–4261. doi: 10.1128/mcb.12.10.4251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kyöstiö S. R., Owens R. A., Weitzman M. D., Antoni B. A., Chejanovsky N., Carter B. J. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels. J Virol. 1994 May;68(5):2947–2957. doi: 10.1128/jvi.68.5.2947-2957.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kyöstiö S. R., Wonderling R. S., Owens R. A. Negative regulation of the adeno-associated virus (AAV) P5 promoter involves both the P5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein. J Virol. 1995 Nov;69(11):6787–6796. doi: 10.1128/jvi.69.11.6787-6796.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Labow M. A., Hermonat P. L., Berns K. I. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. doi: 10.1128/jvi.60.1.251-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laughlin C. A., Jones N., Carter B. J. Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus. J Virol. 1982 Mar;41(3):868–876. doi: 10.1128/jvi.41.3.868-876.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee K. A., Hai T. Y., SivaRaman L., Thimmappaya B., Hurst H. C., Jones N. C., Green M. R. A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8355–8359. doi: 10.1073/pnas.84.23.8355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lorson C., Burger L. R., Mouw M., Pintel D. J. Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1. J Virol. 1996 Feb;70(2):834–842. doi: 10.1128/jvi.70.2.834-842.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McCarty D. M., Christensen M., Muzyczka N. Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein. J Virol. 1991 Jun;65(6):2936–2945. doi: 10.1128/jvi.65.6.2936-2945.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McCarty D. M., Ni T. H., Muzyczka N. Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro. J Virol. 1992 Jul;66(7):4050–4057. doi: 10.1128/jvi.66.7.4050-4057.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McCarty D. M., Pereira D. J., Zolotukhin I., Zhou X., Ryan J. H., Muzyczka N. Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol. 1994 Aug;68(8):4988–4997. doi: 10.1128/jvi.68.8.4988-4997.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McCarty D. M., Ryan J. H., Zolotukhin S., Zhou X., Muzyczka N. Interaction of the adeno-associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat. J Virol. 1994 Aug;68(8):4998–5006. doi: 10.1128/jvi.68.8.4998-5006.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McPherson R. A., Rosenthal L. J., Rose J. A. Human cytomegalovirus completely helps adeno-associated virus replication. Virology. 1985 Nov;147(1):217–222. doi: 10.1016/0042-6822(85)90243-0. [DOI] [PubMed] [Google Scholar]
  38. Mendelson E., Trempe J. P., Carter B. J. Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. J Virol. 1986 Dec;60(3):823–832. doi: 10.1128/jvi.60.3.823-832.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ostrove J. M., Berns K. I. Adenovirus early region 1b gene function required for rescue of latent adeno-associated virus. Virology. 1980 Jul 30;104(2):502–505. doi: 10.1016/0042-6822(80)90354-2. [DOI] [PubMed] [Google Scholar]
  40. Pereira D. J., McCarty D. M., Muzyczka N. The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection. J Virol. 1997 Feb;71(2):1079–1088. doi: 10.1128/jvi.71.2.1079-1088.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pereira D. J., Muzyczka N. The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter. J Virol. 1997 Mar;71(3):1747–1756. doi: 10.1128/jvi.71.3.1747-1756.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pitluk Z. W., Ward D. C. Unusual Sp1-GC box interaction in a parvovirus promoter. J Virol. 1991 Dec;65(12):6661–6670. doi: 10.1128/jvi.65.12.6661-6670.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rhode S. L., 3rd, Richard S. M. Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter. J Virol. 1987 Sep;61(9):2807–2815. doi: 10.1128/jvi.61.9.2807-2815.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Richardson W. D., Westphal H. Adenovirus early gene regulation and the adeno-associated virus helper effect. Curr Top Microbiol Immunol. 1984;109:147–165. doi: 10.1007/978-3-642-69460-8_7. [DOI] [PubMed] [Google Scholar]
  45. Richardson W. D., Westphal H. Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus. J Virol. 1984 Aug;51(2):404–410. doi: 10.1128/jvi.51.2.404-410.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sawadogo M., Roeder R. G. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell. 1985 Nov;43(1):165–175. doi: 10.1016/0092-8674(85)90021-2. [DOI] [PubMed] [Google Scholar]
  47. Shi Y., Seto E., Chang L. S., Shenk T. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein. Cell. 1991 Oct 18;67(2):377–388. doi: 10.1016/0092-8674(91)90189-6. [DOI] [PubMed] [Google Scholar]
  48. Srivastava A., Lusby E. W., Berns K. I. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. doi: 10.1128/jvi.45.2.555-564.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thomson B. J., Weindler F. W., Gray D., Schwaab V., Heilbronn R. Human herpesvirus 6 (HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2) and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression. Virology. 1994 Oct;204(1):304–311. doi: 10.1006/viro.1994.1535. [DOI] [PubMed] [Google Scholar]
  50. Tratschin J. D., Miller I. L., Carter B. J. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol. 1984 Sep;51(3):611–619. doi: 10.1128/jvi.51.3.611-619.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tratschin J. D., Tal J., Carter B. J. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. doi: 10.1128/mcb.6.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Trempe J. P., Mendelson E., Carter B. J. Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli. Virology. 1987 Nov;161(1):18–28. doi: 10.1016/0042-6822(87)90166-8. [DOI] [PubMed] [Google Scholar]
  53. West M. H., Trempe J. P., Tratschin J. D., Carter B. J. Gene expression in adeno-associated virus vectors: the effects of chimeric mRNA structure, helper virus, and adenovirus VA1 RNA. Virology. 1987 Sep;160(1):38–47. doi: 10.1016/0042-6822(87)90041-9. [DOI] [PubMed] [Google Scholar]
  54. Yang Q., Kadam A., Trempe J. P. Mutational analysis of the adeno-associated virus rep gene. J Virol. 1992 Oct;66(10):6058–6069. doi: 10.1128/jvi.66.10.6058-6069.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. van der Vliet P. C., Verrijzer C. P. Bending of DNA by transcription factors. Bioessays. 1993 Jan;15(1):25–32. doi: 10.1002/bies.950150105. [DOI] [PubMed] [Google Scholar]

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

RESOURCES