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. 1996 Jul 23;93(15):7966–7972. doi: 10.1073/pnas.93.15.7966

The recombination signals for adeno-associated virus site-specific integration.

R M Linden 1, E Winocour 1, K I Berns 1
PMCID: PMC38858  PMID: 8755586

Abstract

The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.

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Selected References

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

  1. ATCHISON R. W., CASTO B. C., HAMMON W. M. ADENOVIRUS-ASSOCIATED DEFECTIVE VIRUS PARTICLES. Science. 1965 Aug 13;149(3685):754–756. doi: 10.1126/science.149.3685.754. [DOI] [PubMed] [Google Scholar]
  2. Berns K. I., Linden R. M. The cryptic life style of adeno-associated virus. Bioessays. 1995 Mar;17(3):237–245. doi: 10.1002/bies.950170310. [DOI] [PubMed] [Google Scholar]
  3. Berns K. I., Pinkerton T. C., Thomas G. F., Hoggan M. D. Detection of adeno-associated virus (AAV)-specific nucleotide sequences in DNA isolated from latently infected Detroit 6 cells. Virology. 1975 Dec;68(2):556–560. doi: 10.1016/0042-6822(75)90298-6. [DOI] [PubMed] [Google Scholar]
  4. Buller R. M., Janik J. E., Sebring E. D., Rose J. A. Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J Virol. 1981 Oct;40(1):241–247. doi: 10.1128/jvi.40.1.241-247.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Canfield V., Emanuel J. R., Spickofsky N., Levenson R., Margolskee R. F. Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector. Mol Cell Biol. 1990 Apr;10(4):1367–1372. doi: 10.1128/mcb.10.4.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheung A. K., Hoggan M. D., Hauswirth W. W., Berns K. I. Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol. 1980 Feb;33(2):739–748. doi: 10.1128/jvi.33.2.739-748.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Giraud C., Winocour E., Berns K. I. Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome. J Virol. 1995 Nov;69(11):6917–6924. doi: 10.1128/jvi.69.11.6917-6924.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giraud C., Winocour E., Berns K. I. Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10039–10043. doi: 10.1073/pnas.91.21.10039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Grimm C., Bähler J., Kohli J. M26 recombinational hotspot and physical conversion tract analysis in the ade6 gene of Schizosaccharomyces pombe. Genetics. 1994 Jan;136(1):41–51. doi: 10.1093/genetics/136.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Handa H., Shiroki K., Shimojo H. Complementation of adeno-associated virus growth with temperature-sensitive mutants of human adenovirus types 12 and 5. J Gen Virol. 1975 Nov;29(2):239–242. doi: 10.1099/0022-1317-29-2-239. [DOI] [PubMed] [Google Scholar]
  13. Hauswirth W. W., Berns K. I. Adeno-associated virus DNA replication: nonunit-length molecules. Virology. 1979 Feb;93(1):57–68. doi: 10.1016/0042-6822(79)90275-7. [DOI] [PubMed] [Google Scholar]
  14. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  15. Hoggan M. D., Blacklow N. R., Rowe W. P. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1467–1474. doi: 10.1073/pnas.55.6.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Kotin R. M., Berns K. I. Organization of adeno-associated virus DNA in latently infected Detroit 6 cells. Virology. 1989 Jun;170(2):460–467. doi: 10.1016/0042-6822(89)90437-6. [DOI] [PubMed] [Google Scholar]
  19. Kotin R. M., Linden R. M., Berns K. I. Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 1992 Dec;11(13):5071–5078. doi: 10.1002/j.1460-2075.1992.tb05614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kotin R. M., Menninger J. C., Ward D. C., Berns K. I. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. doi: 10.1016/0888-7543(91)90470-y. [DOI] [PubMed] [Google Scholar]
  21. Kotin R. M. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther. 1994 Jul;5(7):793–801. doi: 10.1089/hum.1994.5.7-793. [DOI] [PubMed] [Google Scholar]
  22. Kotin R. M., Siniscalco M., Samulski R. J., Zhu X. D., Hunter L., Laughlin C. A., McLaughlin S., Muzyczka N., Rocchi M., Berns K. I. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. doi: 10.1073/pnas.87.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis S. M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol. 1994;56:27–150. doi: 10.1016/s0065-2776(08)60450-2. [DOI] [PubMed] [Google Scholar]
  24. Mendelson E., Smith M. G., Miller I. L., Carter B. J. Effect of a viral rep gene on transformation of cells by an adeno-associated virus vector. Virology. 1988 Oct;166(2):612–615. doi: 10.1016/0042-6822(88)90536-3. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Samulski R. J., Zhu X., Xiao X., Brook J. D., Housman D. E., Epstein N., Hunter L. A. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. doi: 10.1002/j.1460-2075.1991.tb04964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schuchert P., Langsford M., Käslin E., Kohli J. A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast. EMBO J. 1991 Aug;10(8):2157–2163. doi: 10.1002/j.1460-2075.1991.tb07750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Snyder R. O., Im D. S., Ni T., Xiao X., Samulski R. J., Muzyczka N. Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein. J Virol. 1993 Oct;67(10):6096–6104. doi: 10.1128/jvi.67.10.6096-6104.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Stamatatos L., Leventis R., Zuckermann M. J., Silvius J. R. Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry. 1988 May 31;27(11):3917–3925. doi: 10.1021/bi00411a005. [DOI] [PubMed] [Google Scholar]
  31. Straus S. E., Sebring E. D., Rose J. A. Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci U S A. 1976 Mar;73(3):742–746. doi: 10.1073/pnas.73.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Urcelay E., Ward P., Wiener S. M., Safer B., Kotin R. M. Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein. J Virol. 1995 Apr;69(4):2038–2046. doi: 10.1128/jvi.69.4.2038-2046.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weindler F. W., Heilbronn R. A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J Virol. 1991 May;65(5):2476–2483. doi: 10.1128/jvi.65.5.2476-2483.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Yakobson B., Hrynko T. A., Peak M. J., Winocour E. Replication of adeno-associated virus in cells irradiated with UV light at 254 nm. J Virol. 1989 Mar;63(3):1023–1030. doi: 10.1128/jvi.63.3.1023-1030.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yakobson B., Koch T., Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol. 1987 Apr;61(4):972–981. doi: 10.1128/jvi.61.4.972-981.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yalkinoglu A. O., Heilbronn R., Bürkle A., Schlehofer J. R., zur Hausen H. DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res. 1988 Jun 1;48(11):3123–3129. [PubMed] [Google Scholar]
  38. Yates J. L., Warren N., Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. 1985 Feb 28-Mar 6Nature. 313(6005):812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]

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