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Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):6787–6796. doi: 10.1128/jvi.69.11.6787-6796.1995

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.

S R Kyöstiö 1, R S Wonderling 1, R A Owens 1
PMCID: PMC189590  PMID: 7474090

Abstract

Transcript levels from the P5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P5 promoter between the P5 TATA box and transcription start site by using 5' and 3' deletions of the P5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P5 RRS DNA. Within the P5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P5 transcript levels by Rep68; one involves Rep68 binding to the P5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS- and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P5 interferes with Rep68 repression of the P5 promoter.

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Selected References

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  1. Antoni B. A., Rabson A. B., Miller I. L., Trempe J. P., Chejanovsky N., Carter B. J. Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells. J Virol. 1991 Jan;65(1):396–404. doi: 10.1128/jvi.65.1.396-404.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
  4. Borowiec J. A., Hurwitz J. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc Natl Acad Sci U S A. 1988 Jan;85(1):64–68. doi: 10.1073/pnas.85.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caswell R., Hagemeier C., Chiou C. J., Hayward G., Kouzarides T., Sinclair J. The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation. J Gen Virol. 1993 Dec;74(Pt 12):2691–2698. doi: 10.1099/0022-1317-74-12-2691. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chejanovsky N., Carter B. J. Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication. J Virol. 1990 Apr;64(4):1764–1770. doi: 10.1128/jvi.64.4.1764-1770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chejanovsky N., Carter B. J. Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor. Virology. 1989 Jul;171(1):239–247. doi: 10.1016/0042-6822(89)90531-x. [DOI] [PubMed] [Google Scholar]
  9. Cherrington J. M., Khoury E. L., Mocarski E. S. Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J Virol. 1991 Feb;65(2):887–896. doi: 10.1128/jvi.65.2.887-896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chiorini J. A., Weitzman M. D., Owens R. A., Urcelay E., Safer B., Kotin R. M. Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli. J Virol. 1994 Feb;68(2):797–804. doi: 10.1128/jvi.68.2.797-804.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chiorini J. A., Wiener S. M., Owens R. A., Kyöstió S. R., Kotin R. M., Safer B. Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats. J Virol. 1994 Nov;68(11):7448–7457. doi: 10.1128/jvi.68.11.7448-7457.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cotmore S. F., Christensen J., Nüesch J. P., Tattersall P. The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3. J Virol. 1995 Mar;69(3):1652–1660. doi: 10.1128/jvi.69.3.1652-1660.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deb S. P., Tegtmeyer P. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication. J Virol. 1987 Dec;61(12):3649–3654. doi: 10.1128/jvi.61.12.3649-3654.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Flotte T. R., Solow R., Owens R. A., Afione S., Zeitlin P. L., Carter B. J. Gene expression from adeno-associated virus vectors in airway epithelial cells. Am J Respir Cell Mol Biol. 1992 Sep;7(3):349–356. doi: 10.1165/ajrcmb/7.3.349. [DOI] [PubMed] [Google Scholar]
  16. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Gustafsson C. M., Hammarsten O., Falkenberg M., Elias P. Herpes simplex virus DNA replication: a spacer sequence directs the ATP-dependent formation of a nucleoprotein complex at oriS. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4629–4633. doi: 10.1073/pnas.91.11.4629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Im D. S., Muzyczka N. Factors that bind to adeno-associated virus terminal repeats. J Virol. 1989 Jul;63(7):3095–3104. doi: 10.1128/jvi.63.7.3095-3104.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Im D. S., Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell. 1990 May 4;61(3):447–457. doi: 10.1016/0092-8674(90)90526-k. [DOI] [PubMed] [Google Scholar]
  22. Jindal H. K., Yong C. B., Wilson G. M., Tam P., Astell C. R. Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions. J Biol Chem. 1994 Feb 4;269(5):3283–3289. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Laughlin C. A., Tratschin J. D., Coon H., Carter B. J. Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene. 1983 Jul;23(1):65–73. doi: 10.1016/0378-1119(83)90217-2. [DOI] [PubMed] [Google Scholar]
  26. Legendre D., Rommelaere J. Terminal regions of the NS-1 protein of the parvovirus minute virus of mice are involved in cytotoxicity and promoter trans inhibition. J Virol. 1992 Oct;66(10):5705–5713. doi: 10.1128/jvi.66.10.5705-5713.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leza M. A., Hearing P. Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element. J Virol. 1988 Aug;62(8):3003–3013. doi: 10.1128/jvi.62.8.3003-3013.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu B., Hermiston T. W., Stinski M. F. A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2. J Virol. 1991 Feb;65(2):897–903. doi: 10.1128/jvi.65.2.897-903.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lorimer H. E., Wang E. H., Prives C. The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides. J Virol. 1991 Feb;65(2):687–699. doi: 10.1128/jvi.65.2.687-699.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lusby E. W., Berns K. I. Mapping of the 5' termini of two adeno-associated virus 2 RNAs in the left half of the genome. J Virol. 1982 Feb;41(2):518–526. doi: 10.1128/jvi.41.2.518-526.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lusky M., Hurwitz J., Seo Y. S. Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site. J Biol Chem. 1993 Jul 25;268(21):15795–15803. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Momoeda M., Wong S., Kawase M., Young N. S., Kajigaya S. A putative nucleoside triphosphate-binding domain in the nonstructural protein of B19 parvovirus is required for cytotoxicity. J Virol. 1994 Dec;68(12):8443–8446. doi: 10.1128/jvi.68.12.8443-8446.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Muller M. T. Binding of the herpes simplex virus immediate-early gene product ICP4 to its own transcription start site. J Virol. 1987 Mar;61(3):858–865. doi: 10.1128/jvi.61.3.858-865.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oelze I., Rittner K., Sczakiel G. Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions. J Virol. 1994 Feb;68(2):1229–1233. doi: 10.1128/jvi.68.2.1229-1233.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Owens R. A., Weitzman M. D., Kyöstiö S. R., Carter B. J. Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins. J Virol. 1993 Feb;67(2):997–1005. doi: 10.1128/jvi.67.2.997-1005.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rittner K., Heilbronn R., Kleinschmidt J. A., Sczakiel G. Adeno-associated virus type 2-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) replication: involvement of p78rep/p68rep and the HIV-1 long terminal repeat. J Gen Virol. 1992 Nov;73(Pt 11):2977–2981. doi: 10.1099/0022-1317-73-11-2977. [DOI] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seto E., Shi Y., Shenk T. YY1 is an initiator sequence-binding protein that directs and activates transcription in vitro. Nature. 1991 Nov 21;354(6350):241–245. doi: 10.1038/354241a0. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Smith C. A., Bates P., Rivera-Gonzalez R., Gu B., DeLuca N. A. ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB. J Virol. 1993 Aug;67(8):4676–4687. doi: 10.1128/jvi.67.8.4676-4687.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Snyder R. O., Im D. S., Muzyczka N. Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome. J Virol. 1990 Dec;64(12):6204–6213. doi: 10.1128/jvi.64.12.6204-6213.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. 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]
  49. Trempe J. P., Carter B. J. Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein. J Virol. 1988 Sep;62(9):3356–3363. doi: 10.1128/jvi.62.9.3356-3363.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Trempe J. P., Carter B. J. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation. J Virol. 1988 Jan;62(1):68–74. doi: 10.1128/jvi.62.1.68-74.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Weitzman M. D., Kyöstiö S. R., Kotin R. M., Owens R. A. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5808–5812. doi: 10.1073/pnas.91.13.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wonderling R. S., Kyöstiö S. R., Owens R. A. A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. J Virol. 1995 Jun;69(6):3542–3548. doi: 10.1128/jvi.69.6.3542-3548.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu J., Jupp R., Stenberg R. M., Nelson J. A., Ghazal P. Site-specific inhibition of RNA polymerase II preinitiation complex assembly by human cytomegalovirus IE86 protein. J Virol. 1993 Dec;67(12):7547–7555. doi: 10.1128/jvi.67.12.7547-7555.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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