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. 1997 Apr;71(4):3299–3306. doi: 10.1128/jvi.71.4.3299-3306.1997

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

C Balagúe 1, M Kalla 1, W W Zhang 1
PMCID: PMC191468  PMID: 9060699

Abstract

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.

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Selected References

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  1. Afione S. A., Conrad C. K., Kearns W. G., Chunduru S., Adams R., Reynolds T. C., Guggino W. B., Cutting G. R., Carter B. J., Flotte T. R. In vivo model of adeno-associated virus vector persistence and rescue. J Virol. 1996 May;70(5):3235–3241. doi: 10.1128/jvi.70.5.3235-3241.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashktorab H., Srivastava A. Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA. J Virol. 1989 Jul;63(7):3034–3039. doi: 10.1128/jvi.63.7.3034-3039.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Flotte T. R., Afione S. A., Zeitlin P. L. Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol. 1994 Nov;11(5):517–521. doi: 10.1165/ajrcmb.11.5.7946381. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Gottlieb J., Muzyczka N. In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences. Mol Cell Biol. 1988 Jun;8(6):2513–2522. doi: 10.1128/mcb.8.6.2513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hong G., Ward P., Berns K. I. Intermediates of adeno-associated virus DNA replication in vitro. J Virol. 1994 Mar;68(3):2011–2015. doi: 10.1128/jvi.68.3.2011-2015.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Laughlin C. A., Cardellichio C. B., Coon H. C. Latent infection of KB cells with adeno-associated virus type 2. J Virol. 1986 Nov;60(2):515–524. doi: 10.1128/jvi.60.2.515-524.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Linden R. M., Winocour E., Berns K. I. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966–7972. doi: 10.1073/pnas.93.15.7966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McLaughlin S. K., Collis P., Hermonat P. L., Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6):1963–1973. doi: 10.1128/jvi.62.6.1963-1973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Philip R., Brunette E., Kilinski L., Murugesh D., McNally M. A., Ucar K., Rosenblatt J., Okarma T. B., Lebkowski J. S. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes. Mol Cell Biol. 1994 Apr;14(4):2411–2418. doi: 10.1128/mcb.14.4.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Russell D. W., Miller A. D., Alexander I. E. Adeno-associated virus vectors preferentially transduce cells in S phase. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8915–8919. doi: 10.1073/pnas.91.19.8915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Samulski R. J. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. doi: 10.1016/s0959-437x(05)80344-2. [DOI] [PubMed] [Google Scholar]
  22. Samulski R. J., Chang L. S., 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. doi: 10.1128/jvi.61.10.3096-3101.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Shelling A. N., Smith M. G. Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene. Gene Ther. 1994 May;1(3):165–169. [PubMed] [Google Scholar]
  25. Snyder R. O., Samulski R. J., Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends. Cell. 1990 Jan 12;60(1):105–113. doi: 10.1016/0092-8674(90)90720-y. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Walsh C. E., Liu J. M., Xiao X., Young N. S., Nienhuis A. W., Samulski R. J. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7257–7261. doi: 10.1073/pnas.89.15.7257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Walz C., Schlehofer J. R. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17. J Virol. 1992 May;66(5):2990–3002. doi: 10.1128/jvi.66.5.2990-3002.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Xiao X., Li J., Samulski R. J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol. 1996 Nov;70(11):8098–8108. doi: 10.1128/jvi.70.11.8098-8108.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang Q., Chen F., Trempe J. P. Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol. 1994 Aug;68(8):4847–4856. doi: 10.1128/jvi.68.8.4847-4856.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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