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. 1991 Dec;10(12):3941–3950. doi: 10.1002/j.1460-2075.1991.tb04964.x

Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

R J Samulski 1, X Zhu 1, X Xiao 1, J D Brook 1, D E Housman 1, N Epstein 1, L A Hunter 1
PMCID: PMC453134  PMID: 1657596

Abstract

A key feature in adeno-associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein--DNA binding method we isolated AAV--cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus-free parental cell line, and also co-migrated with AAV-specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4-ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non-pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site-specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.

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Selected References

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  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. 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. Berger R., Bloomfield C. D., Sutherland G. R. Report of the Committee on Chromosome Rearrangements in Neoplasia and on Fragile Sites. Cytogenet Cell Genet. 1985;40(1-4):490–535. doi: 10.1159/000132181. [DOI] [PubMed] [Google Scholar]
  4. Bergeron D., Poliquin L., Kozak C. A., Rassart E. Identification of a common viral integration region in Cas-Br-E murine leukemia virus-induced non-T-, non-B-cell lymphomas. J Virol. 1991 Jan;65(1):7–15. doi: 10.1128/jvi.65.1.7-15.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berns A., Breuer M., Verbeek S., van Lohuizen M. Transgenic mice as a means to study synergism between oncogenes. Int J Cancer Suppl. 1989;4:22–25. doi: 10.1002/ijc.2910440706. [DOI] [PubMed] [Google Scholar]
  6. Bohenzky R. A., LeFebvre R. B., Berns K. I. Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat. Virology. 1988 Oct;166(2):316–327. doi: 10.1016/0042-6822(88)90502-8. [DOI] [PubMed] [Google Scholar]
  7. Brook J. D., Harley H. G., Walsh K. V., Rundle S. A., Siciliano M. J., Harper P. S., Shaw D. J. Identification of new DNA markers close to the myotonic dystrophy locus. J Med Genet. 1991 Feb;28(2):84–88. doi: 10.1136/jmg.28.2.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Casey G., Smith R., McGillivray D., Peters G., Dickson C. Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene. Mol Cell Biol. 1986 Feb;6(2):502–510. doi: 10.1128/mcb.6.2.502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Cohen J. C., Murphey-Corb M. Targeted integration of baboon endogenous virus in the BEVI locus on human chromosome 6. Nature. 1983 Jan 13;301(5896):129–132. doi: 10.1038/301129a0. [DOI] [PubMed] [Google Scholar]
  13. Deiss V., Tratschin J. D., Weitz M., Siegl G. Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini. Virology. 1990 Mar;175(1):247–254. doi: 10.1016/0042-6822(90)90205-6. [DOI] [PubMed] [Google Scholar]
  14. Epstein N. D., Karlsson S., O'Brien S., Modi W., Moulton A., Nienhuis A. W. A new moderately repetitive DNA sequence family of novel organization. Nucleic Acids Res. 1987 Mar 11;15(5):2327–2341. doi: 10.1093/nar/15.5.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  16. Gallahan D., Kozak C., Callahan R. A new common integration region (int-3) for mouse mammary tumor virus on mouse chromosome 17. J Virol. 1987 Jan;61(1):218–220. doi: 10.1128/jvi.61.1.218-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garcia M., Wellinger R., Vessaz A., Diggelmann H. A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas. EMBO J. 1986 Jan;5(1):127–134. doi: 10.1002/j.1460-2075.1986.tb04186.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geissler E. N., Liao M., Brook J. D., Martin F. H., Zsebo K. M., Housman D. E., Galli S. J. Stem cell factor (SCF), a novel hematopoietic growth factor and ligand for c-kit tyrosine kinase receptor, maps on human chromosome 12 between 12q14.3 and 12qter. Somat Cell Mol Genet. 1991 Mar;17(2):207–214. doi: 10.1007/BF01232978. [DOI] [PubMed] [Google Scholar]
  19. Grandgenett D. P., Mumm S. R. Unraveling retrovirus integration. Cell. 1990 Jan 12;60(1):3–4. doi: 10.1016/0092-8674(90)90707-l. [DOI] [PubMed] [Google Scholar]
  20. Handa H., Shiroki K., Shimojo H. Establishment and characterization of KB cell lines latently infected with adeno-associated virus type 1. Virology. 1977 Oct 1;82(1):84–92. doi: 10.1016/0042-6822(77)90034-4. [DOI] [PubMed] [Google Scholar]
  21. Hermonat P. L., Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6466–6470. doi: 10.1073/pnas.81.20.6466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang L. H., Silberman J., Rothschild H., Cohen J. C. Replication of baboon endogenous virus in human cells. Kinetics of DNA synthesis and integration. J Biol Chem. 1989 May 25;264(15):8811–8814. [PubMed] [Google Scholar]
  23. Hughes S. H., Shank P. R., Spector D. H., Kung H. J., Bishop J. M., Varmus H. E., Vogt P. K., Breitman M. L. Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites. Cell. 1978 Dec;15(4):1397–1410. doi: 10.1016/0092-8674(78)90064-8. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Johnson A. D., Poteete A. R., Lauer G., Sauer R. T., Ackers G. K., Ptashne M. lambda Repressor and cro--components of an efficient molecular switch. Nature. 1981 Nov 19;294(5838):217–223. doi: 10.1038/294217a0. [DOI] [PubMed] [Google Scholar]
  27. Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods. 1981;43(3):349–350. doi: 10.1016/0022-1759(81)90183-6. [DOI] [PubMed] [Google Scholar]
  28. Jourdan M., Jousset F. X., Gervais M., Skory S., Bergoin M., Dumas B. Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis. Virology. 1990 Nov;179(1):403–409. doi: 10.1016/0042-6822(90)90308-e. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. LaFace D., Hermonat P., Wakeland E., Peck A. Gene transfer into hematopoietic progenitor cells mediated by an adeno-associated virus vector. Virology. 1988 Feb;162(2):483–486. doi: 10.1016/0042-6822(88)90491-6. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [DOI] [PubMed] [Google Scholar]
  34. Lemons R. S., Nash W. G., O'Brien S. J., Benveniste R. E., Sherr C. J. A gene (Bevi) on human chromosome 6 is an integration site for baboon type C DNA provirus in human cells. Cell. 1978 Aug;14(4):995–1005. doi: 10.1016/0092-8674(78)90353-7. [DOI] [PubMed] [Google Scholar]
  35. Lemons R. S., O'Brien S. J., Sherr C. J. A new genetic locus, Bevi, on human chromosome 6 which controls the replication of baboon type C virus in human cells. Cell. 1977 Sep;12(1):251–262. doi: 10.1016/0092-8674(77)90203-3. [DOI] [PubMed] [Google Scholar]
  36. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Merchlinsky M. J., Tattersall P. J., Leary J. J., Cotmore S. F., Gardiner E. M., Ward D. C. Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice. J Virol. 1983 Jul;47(1):227–232. doi: 10.1128/jvi.47.1.227-232.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nusse R., Varmus H. E. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell. 1982 Nov;31(1):99–109. doi: 10.1016/0092-8674(82)90409-3. [DOI] [PubMed] [Google Scholar]
  41. Peters G., Lee A. E., Dickson C. Concerted activation of two potential proto-oncogenes in carcinomas induced by mouse mammary tumour virus. Nature. 1986 Apr 17;320(6063):628–631. doi: 10.1038/320628a0. [DOI] [PubMed] [Google Scholar]
  42. Popescu N. C., Amsbaugh S. C., DiPaolo J. A. Human papillomavirus type 18 DNA is integrated at a single chromosome site in cervical carcinoma cell line SW756. J Virol. 1987 May;61(5):1682–1685. doi: 10.1128/jvi.61.5.1682-1685.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Popescu N. C., DiPaolo J. A., Amsbaugh S. C. Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes. Cytogenet Cell Genet. 1987;44(1):58–62. doi: 10.1159/000132342. [DOI] [PubMed] [Google Scholar]
  44. Ringold G. M., Shank P. R., Varmus H. E., Ring J., Yamamoto K. R. Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells. Proc Natl Acad Sci U S A. 1979 Feb;76(2):665–669. doi: 10.1073/pnas.76.2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rohdewohld H., Weiher H., Reik W., Jaenisch R., Breindl M. Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites. J Virol. 1987 Feb;61(2):336–343. doi: 10.1128/jvi.61.2.336-343.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Romani M., De Ambrosis A., Alhadeff B., Purrello M., Gluzman Y., Siniscalco M. Preferential integration of the Ad5/SV40 hybrid virus at the highly recombinogenic human chromosomal site 1p36. Gene. 1990 Nov 15;95(2):231–241. doi: 10.1016/0378-1119(90)90366-y. [DOI] [PubMed] [Google Scholar]
  47. Samulski R. J., Berns K. I., 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. doi: 10.1073/pnas.79.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Samulski R. J., Chang L. S., 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. doi: 10.1128/jvi.63.9.3822-3828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Samulski R. J., Srivastava A., Berns K. I., Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983 May;33(1):135–143. doi: 10.1016/0092-8674(83)90342-2. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Scherdin U., Rhodes K., Breindl M. Transcriptionally active genome regions are preferred targets for retrovirus integration. J Virol. 1990 Feb;64(2):907–912. doi: 10.1128/jvi.64.2.907-912.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shih C. C., Stoye J. P., Coffin J. M. Highly preferred targets for retrovirus integration. Cell. 1988 May 20;53(4):531–537. doi: 10.1016/0092-8674(88)90569-7. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Srivastava C. H., Samulski R. J., Lu L., Larsen S. H., Srivastava A. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8078–8082. doi: 10.1073/pnas.86.20.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Steffen D., Weinberg R. A. The integrated genome of murine leukemia virus. Cell. 1978 Nov;15(3):1003–1010. doi: 10.1016/0092-8674(78)90284-2. [DOI] [PubMed] [Google Scholar]
  58. Sutherland G. R., Mattei J. F. Report of the committee on cytogenetic markers. Cytogenet Cell Genet. 1987;46(1-4):316–324. doi: 10.1159/000132482. [DOI] [PubMed] [Google Scholar]
  59. Tratschin J. D., Miller I. L., Smith M. G., Carter B. J. Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells. Mol Cell Biol. 1985 Nov;5(11):3251–3260. doi: 10.1128/mcb.5.11.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vijaya S., Steffen D. L., Robinson H. L. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin. J Virol. 1986 Nov;60(2):683–692. doi: 10.1128/jvi.60.2.683-692.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Warburton D., Gersen S., Yu M. T., Jackson C., Handelin B., Housman D. Monochromosomal rodent-human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker. Genomics. 1990 Feb;6(2):358–366. doi: 10.1016/0888-7543(90)90577-h. [DOI] [PubMed] [Google Scholar]
  62. van Lohuizen M., Breuer M., Berns A. N-myc is frequently activated by proviral insertion in MuLV-induced T cell lymphomas. EMBO J. 1989 Jan;8(1):133–136. doi: 10.1002/j.1460-2075.1989.tb03357.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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