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J Virol. Oct 1997; 71(10): 7951–7959.
PMCID: PMC192153

Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

Abstract

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adair GM, Nairn RS, Wilson JH, Seidman MM, Brotherman KA, MacKinnon C, Scheerer JB. Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4574–4578. [PMC free article] [PubMed]
  • Afione SA, Conrad CK, Kearns WG, Chunduru S, Adams R, Reynolds TC, Guggino WB, Cutting GR, Carter BJ, Flotte TR. In vivo model of adeno-associated virus vector persistence and rescue. J Virol. 1996 May;70(5):3235–3241. [PMC free article] [PubMed]
  • Beaton A, Palumbo P, Berns KI. 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. [PMC free article] [PubMed]
  • Berns KI. Parvovirus replication. Microbiol Rev. 1990 Sep;54(3):316–329. [PMC free article] [PubMed]
  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed]
  • Boshart M, Weber F, Jahn G, Dorsch-Häsler K, Fleckenstein B, Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. [PubMed]
  • Britten RJ, Baron WF, Stout DB, Davidson EH. Sources and evolution of human Alu repeated sequences. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4770–4774. [PMC free article] [PubMed]
  • Chang LS, 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. [PMC free article] [PubMed]
  • Cheung AK, Hoggan MD, Hauswirth WW, Berns KI. 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. [PMC free article] [PubMed]
  • Chiorini JA, Weitzman MD, Owens RA, Urcelay E, Safer B, Kotin RM. 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. [PMC free article] [PubMed]
  • Chiorini JA, Yang L, Safer B, Kotin RM. Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection. J Virol. 1995 Nov;69(11):7334–7338. [PMC free article] [PubMed]
  • Doetschman T, Maeda N, Smithies O. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8583–8587. [PMC free article] [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • Giraud C, Winocour E, Berns KI. Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome. J Virol. 1995 Nov;69(11):6917–6924. [PMC free article] [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hauswirth WW, Berns KI. Adeno-associated virus DNA replication: nonunit-length molecules. Virology. 1979 Feb;93(1):57–68. [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Hölscher C, Hörer M, Kleinschmidt JA, Zentgraf H, Bürkle A, Heilbronn R. Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants. J Virol. 1994 Nov;68(11):7169–7177. [PMC free article] [PubMed]
  • Im DS, 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. [PubMed]
  • Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR. Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther. 1996 Sep;3(9):748–755. [PubMed]
  • Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14082–14087. [PMC free article] [PubMed]
  • Kotin RM. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther. 1994 Jul;5(7):793–801. [PubMed]
  • Kotin RM, Berns KI. Organization of adeno-associated virus DNA in latently infected Detroit 6 cells. Virology. 1989 Jun;170(2):460–467. [PubMed]
  • Kotin RM, Linden RM, Berns KI. 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. [PMC free article] [PubMed]
  • Kotin RM, Menninger JC, Ward DC, Berns KI. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. [PubMed]
  • Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. [PMC free article] [PubMed]
  • Labow MA, Graf LH, Jr, Berns KI. Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol. 1987 Apr;7(4):1320–1325. [PMC free article] [PubMed]
  • Labow MA, Hermonat PL, Berns KI. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. [PMC free article] [PubMed]
  • Laughlin CA, Cardellichio CB, Coon HC. Latent infection of KB cells with adeno-associated virus type 2. J Virol. 1986 Nov;60(2):515–524. [PMC free article] [PubMed]
  • Laughlin CA, Tratschin JD, Coon H, Carter BJ. Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene. 1983 Jul;23(1):65–73. [PubMed]
  • Linden RM, Winocour E, Berns KI. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966–7972. [PMC free article] [PubMed]
  • McCarty DM, Pereira DJ, Zolotukhin I, Zhou X, Ryan JH, Muzyczka N. Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol. 1994 Aug;68(8):4988–4997. [PMC free article] [PubMed]
  • McLaughlin SK, Collis P, Hermonat PL, Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6):1963–1973. [PMC free article] [PubMed]
  • Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. [PubMed]
  • Ni TH, Zhou X, McCarty DM, Zolotukhin I, Muzyczka N. In vitro replication of adeno-associated virus DNA. J Virol. 1994 Feb;68(2):1128–1138. [PMC free article] [PubMed]
  • O'Gorman S, Fox DT, Wahl GM. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991 Mar 15;251(4999):1351–1355. [PubMed]
  • Owens RA, Trempe JP, Chejanovsky N, Carter BJ. Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin. Virology. 1991 Sep;184(1):14–22. [PubMed]
  • Owens RA, Weitzman MD, Kyöstiö SR, Carter BJ. Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins. J Virol. 1993 Feb;67(2):997–1005. [PMC free article] [PubMed]
  • Samulski RJ, Berns KI, Tan M, Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. [PMC free article] [PubMed]
  • Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. [PMC free article] [PubMed]
  • Samulski RJ, Chang LS, Shenk T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol. 1987 Oct;61(10):3096–3101. [PMC free article] [PubMed]
  • Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. [PMC free article] [PubMed]
  • Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. [PMC free article] [PubMed]
  • Shelling AN, Smith MG. Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene. Gene Ther. 1994 May;1(3):165–169. [PubMed]
  • Snyder RO, Im DS, Ni T, Xiao X, Samulski RJ, 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. [PMC free article] [PubMed]
  • Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. [PMC free article] [PubMed]
  • Stokke T, Collins C, Kuo WL, Kowbel D, Shadravan F, Tanner M, Kallioniemi A, Kallioniemi OP, Pinkel D, Deaven L, et al. A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis. Genomics. 1995 Mar 1;26(1):134–137. [PubMed]
  • Straus SE, Sebring ED, Rose JA. 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. [PMC free article] [PubMed]
  • Tratschin JD, Miller IL, Carter BJ. 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. [PMC free article] [PubMed]
  • Weitzman MD, Kyöstiö SR, Kotin RM, Owens RA. 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. [PMC free article] [PubMed]
  • Wigler M, Pellicer A, Silverstein S, Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. [PubMed]
  • Wonderling RS, Owens RA. The Rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene. J Virol. 1996 Jul;70(7):4783–4786. [PMC free article] [PubMed]

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