1887

Abstract

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV inverted terminal repeat (ITR)–chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR–chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.18726-0
2003-01-01
2020-01-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/84/1/vir840133.html?itemId=/content/journal/jgv/10.1099/vir.0.18726-0&mimeType=html&fmt=ahah

References

  1. Berns, K. I. ( 1996; ). Parvoviridae: The viruses and their replication. In Fields Virology, 3rd edn, pp. 2173–2197. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  2. Giraud, C., Winocour, E. & Berns, K. I. ( 1994; ). Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence. Proc Natl Acad Sci U S A 91, 10039–10043.[CrossRef]
    [Google Scholar]
  3. Giraud, C., Winocour, E. & Berns, K. I. ( 1995; ). Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome. J Virol 69, 6917–6924.
    [Google Scholar]
  4. Hüser, D., Weger, S. & Heilbronn, R. ( 2002; ). Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR. J Virol 76, 7554–7559.[CrossRef]
    [Google Scholar]
  5. Im, D.-S. & Muzyczka, N. ( 1990; ). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with helicase activity. Cell 61, 447–457.[CrossRef]
    [Google Scholar]
  6. Kotin, R. M., Siniscalco, M., Samulski, R. J. & 7 other authors ( 1990; ). Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 87, 2211–2215.[CrossRef]
    [Google Scholar]
  7. Kotin, R. M., Linden, R. M. & Berns, K. I. ( 1992; ). Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J 11, 5071–5078.
    [Google Scholar]
  8. Lamartina, S., Sporeno, E., Fattori, E. & Toniatti, C. ( 2000; ). Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment. J Virol 74, 7671–7677.[CrossRef]
    [Google Scholar]
  9. Linden, R. M., Ward, P., Giraud, C., Winocour, E. & Berns, K. I. ( 1996a; ). Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 93, 11288–11294.[CrossRef]
    [Google Scholar]
  10. Linden, R. M., Winocour, E. & Berns, K. I. ( 1996b; ). The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A 93, 7966–7972.[CrossRef]
    [Google Scholar]
  11. Macville, M., Schrock, E., Padilla-Nash, H., Keck, C., Ghadimi, B. M., Zimonjic, D., Popescu, N. & Ried, T. ( 1999; ). Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res 59, 141–150.
    [Google Scholar]
  12. Meneses, P., Berns, K. I. & Winocour, E. ( 2000; ). DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system. J Virol 74, 6213–6216.[CrossRef]
    [Google Scholar]
  13. Miller, D. G., Rutledge, E. A. & Russell, D. W. ( 2002; ). Chromosomal effects of adeno-associated virus vector integration. Nat Genet 30, 147–148.[CrossRef]
    [Google Scholar]
  14. Muzyczka, N. & Berns, K. I. ( 2001; ). Parvoviridae: The viruses and their replication. In Fields Virology, 4th edn, pp. 2327–2359. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
  15. Palombo, F., Monciotti, A., Recchia, A., Cortese, R., Ciliberto, G. & La Monica, N. ( 1998; ). Site-specific integration in mammalian cells mediated by a new hybrid baculovirus–adeno-associated virus vector. J Virol 72, 5025–5034.
    [Google Scholar]
  16. Pieroni, L., Fipaldini, C., Monciotti, A. & 7 other authors ( 1998; ). Targeted integration of adeno-associated virus-derived plasmids in transfected human cells. Virology 249, 249–259.[CrossRef]
    [Google Scholar]
  17. Recchia, A., Parks, R. J., Lamartina, S. & 8 other authors ( 1999; ). Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proc Natl Acad Sci U S A 96, 2615–2620.[CrossRef]
    [Google Scholar]
  18. Rizzuto, G., Gorgoni, B., Cappelletti, M. & 9 other authors ( 1999; ). Development of animal models for adeno-associated virus site-specific integration. J Virol 73, 2517–2526.
    [Google Scholar]
  19. Samulski, R. J., Zhu, X., Xiao, X., Brook, J. D., Housman, D. E., Epstein, N. & Hunter, L. A. ( 1991; ). Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 10, 3941–3950; erratum 11, 1228.
    [Google Scholar]
  20. Snyder, R. O., Im, D.-S., Ni, T., Xiao, X., Samulski, R. J. & Muzyczka, N. ( 1993; ). Features of the adeno-associated virus origin involved in substrate recognition by the viral rep protein. J Virol 67, 6096–6104.
    [Google Scholar]
  21. Srivastava, A., Lusby, E. W. & Berns, K. I. ( 1983; ). Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol 45, 555–564.
    [Google Scholar]
  22. Tsunoda, H., Hayakawa, T., Sakuragawa, N. & Koyama, H. ( 2000; ). Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells. Virology 268, 391–401.[CrossRef]
    [Google Scholar]
  23. Weitzman, M. D., Kyöstiö, S. R. M., Kotin, R. M. & Owens, R. A. ( 1994; ). 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 91, 5808–5812.[CrossRef]
    [Google Scholar]
  24. Yang, C. C., Xiao, X., Zhu, X., Ansardi, D. C., Epstein, N. D., Frey, M. R., Matera, A. G. & Samulski, R. J. ( 1997; ). Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J Virol 71, 9231–9247.
    [Google Scholar]
  25. Young, S. M., Jr & Samulski, R. J. ( 2001; ). Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat. Proc Natl Acad Sci U S A 98, 13525–13530.[CrossRef]
    [Google Scholar]
  26. Young, S. M., Jr., McCarty, D. M., Degtyareva, N. & Samulski, R. J. ( 2000; ). Roles of adeno-associated virus Rep protein and human chromosome 19 in site-specific recombination. J Virol 74, 3953–3966.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.18726-0
Loading
/content/journal/jgv/10.1099/vir.0.18726-0
Loading

Data & Media loading...

Most cited articles

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error