1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi, Tochigi 329-0498, Japan
2 CREST, Japan Science and Technology Corporation (JST), Tochigi 329-0498, Japan
3 Department of Intractable Diseases, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan
4 Department of Virology, Jichi Medical School, Tochigi 329-0498, Japan
Correspondence
Masashi Urabe (at Division of Genetic Therapeutics)
murabe{at}jichi.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
Each end of the AAV genome consists of inverted terminal repeats (ITRs), which are required in cis for AAVS1-specific integration. The AAV rep gene encodes four overlapping non-structural proteins, Rep78, Rep68, Rep52 and Rep40, while the cap gene encodes structural Cap proteins. The unspliced and spliced transcripts from the p5 promoter encode Rep78 and Rep68. Either Rep78 or Rep68 plays a key role in AAVS1-specific integration, binding ITRs (Im & Muzyczka, 1989) and AAVS1 (Weitzman et al., 1994
) via tandem repeats of the GAGC tetramer (McCarty et al., 1994
). The mechanism of AAVS1-specific integration of AAV has not been elucidated fully. However, a model whereby integration proceeds via a circular intermediate of the AAV genome by a deletion-substitution mechanism has been proposed (Dyall & Berns, 1998
; Linden et al., 1996
).
A structural difference between Rep78 and Rep68 is that Rep78 possesses a zinc finger-like motif at its carboxyl terminus. Both Rep proteins share essentially the same functions: strand-specific DNA binding (Im & Muzyczka, 1989), site-specific nicking and ATP-dependent helicase activity (Im & Muzyczka, 1990
). Either Rep protein alone is sufficient for replication of the AAV genome (Hölscher et al., 1994
) and for AAVS1-specific integration (Surosky et al., 1997
). The multifunctional Rep proteins inhibit cellular transformation by heterologous genes (Labow et al., 1987
; Yang et al., 1992
) and suppress heterologous promoters, including the c-fos, c-myc, H-ras and LTR of human immunodeficiency virus type 1 (HIV-1) (Hermonat, 1991
, 1994
; Oelze et al., 1994
). The Rep proteins also modulate cell cycle-regulating proteins (Hermanns et al., 1997
). These results indicate that overexpression of Rep proteins has negative effects on cells and is, on occasion, lethal to cells.
AAV vectors lacking the rep gene fail to integrate into AAVS1, showing apparent random integration into the host chromosomal DNA (Kearns et al., 1996). A non-viral plasmid-based system capable of integrating a transgene specifically into AAVS1 has been described; this was achieved by transferring the transgene flanked by the ITRs with transient expression of Rep78 or Rep68 (Balagué et al., 1997
; Pieroni et al., 1998
; Shelling & Smith, 1994
; Surosky et al., 1997
; Tsunoda et al., 2000
). Thus, this system is safer than integrating retrovirus and AAV vectors randomly. A strategy utilizing two plasmids, one harbouring the transgene cassette between the ITR sequences and the other for Rep expression, allows only the transgene plasmid to integrate into the AAVS1 locus (Surosky et al., 1997
). This method successfully introduced the transgene into AAVS1 in haematopoietic K562 cells (Kogure et al., 2001
).
The frequency of AAVS1-specific integration by the plasmid-based methods has differed among studies. Shelling & Smith (1994) reported that 9 of 12 cell clones (75 %) obtained by transfecting HeLa or 293 cells with an AAV vector plasmid on which the Neo gene was placed under the control of the p40 promoter, the original promoter for Cap proteins, had rearranged AAVS1 and mentioned that approximately 50 % of the rearranged bands also hybridized to an AAV probe. Another strategy using one plasmid on which both a Rep cassette and an ITR-flanked transgene cassette were placed has targeted the transgene to AAVS1 in 6 of 21 (29 %) 293 cell clones (Balagué et al., 1997
). Similar methods applied to other cell lines, HeLa and Huh-7 cells, have been able to insert the transgene to AAVS1 in up to 20 % of clones (Lamartina et al., 1998
; Pieroni et al., 1998
). All the studies mentioned here used a one plasmid system and the p5 promoter for Rep expression.
Aiming at increasing the frequency of AAVS1-directed integration, we first examined whether AAVS1-specific integration depended on the levels of Rep protein expressed in cells. To control the expression of the cytotoxic Rep proteins, we chose to vary the amount of Rep plasmid DNA. 293 cells were transfected using the calcium phosphate precipitation method with 2, 0·4, 0·2, 0·04, 0·02 or 0 µg pCMVR78, which expresses Rep78 under the control of the CMV promoter (Surosky et al., 1997), and 2 µg pWNeo (Rep : Neo ratio of 1, 0·2, 0·1, 0·02, 0·01 or 0). pWNeo bears a Neo gene under the control of the CMV promoter between the ITRs. To monitor the amount of plasmid DNA incorporated, extrachromosomal DNA was analysed by Southern blot with a plasmid backbone probe (Fig. 1
a). As the amount of Rep plasmid decreased, signal intensities corresponding to pCMVR78 decreased gradually, whereas those corresponding to pWNeo changed little, indicating that the amount of plasmid DNA incorporated into the cells correlated with that used for transfection. Western analysis of the transfected 293 cells confirmed that the expression level of the Rep protein was a function of the amount of pCMVR78 (Fig. 1b
).
|
|
Fig. 2 shows Southern blot analysis of representative clones with the Neo gene at AAVS1. Fig. 2(a, b)
is the HindIII- or EcoRV-digest probed with an AAVS1-specific probe (upper panel) or a Neo probe (lower panel). Each clone presented here has an upshifted band(s) other than a basal band (arrow). Common bands that hybridized to both AAVS1 and Neo probes are indicated by arrowheads. Fluorescent in situ hybridization (FISH) analysis confirmed the integration of the Neo gene into chromosome 19 in 11 of 12 clones. A representative chromosomal analysis is shown in Fig. 2(c)
. The 293 cells used in the present study have four copies of chromosome 19 labelled with Cy-3-conjugated chromosome 19-specific probe (arrowheads). The left panel shows a metaphase spread of clone C6/6. Fluorescein Neo signals are localized to chromosome 19 and another unidentified site (arrows). In the right panel showing analysis of clone C6/18, one chromosome 19 harbours the Neo signals at its terminal portion.
|
The 293 cells used in the present study have four copies of chromosome 19. Southern blot analysis showed that some clones had more than three upshifted bands besides a basal band. We used a relatively large probe (3·0 kb) for detecting AAVS1 bands. It is possible that Rep-mediated disruption of the AAVS1 region can produce the multiple bands hybridizing to the AAVS1 probe. Another explanation is as follows: at 24 h post-transfection, we replated transfected cells to isolate clones derived from single cells. At this time-point, the Rep protein was still being expressed in cells and an additional integration event might occur in some cells after cell division.
Lamartina et al. (1998) reported no apparent difference between Rep78 and Rep68 in the ability to deliver foreign DNA to AAVS1 in HeLa cells. Several studies have reported the functional differences between Rep78 and Rep68. Rep68 is more efficient in processing dimers to monomer duplex DNA and possesses a stronger nicking activity (Ni et al., 1994
, 1998
), while the helicase activity of Rep78 is stronger (Wollscheid et al., 1997
). The differential effects of Rep78 and Rep68 on the p5, p19 and p40 promoters were described (Weger et al., 1997
). In addition, Rep78 inhibits CREB-dependent transcription by interacting with protein kinase (Chiorini et al., 1998
; Di Pasquale & Stacey, 1998
). None of these findings explains why Rep78 appears to cause more abortive integration. Rep68 may be more suitable for the AAVS1-targeted integration system. To confirm the usefulness of Rep68 in the AAVS1-targeted integration system, further analysis of a larger population of cell clones would be required. Also, the exact functions of the Rep protein in AAVS1-specific integration should be elucidated.
The results presented here have important implications for developing an AAVS1-directed integration system as well as for understanding the mechanism of AAVS1-specific integration by the Rep proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Berns, K. I. & Giraud, C. (1996). Biology of adeno-associated virus. Curr Top Microbiol Immunol 218, 123.[Medline]
Cheung, A. K., Hoggan, M. D., Hauswirth, W. W. & Berns, K. I. (1980). Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol 33, 739748.[Medline]
Chiorini, J. A., Zimmermann, B., Yang, L., Smith, R. H., Ahearn, A., Herberg, F. & Kotin, R. M. (1998). Inhibition of PrKX, a novel protein kinase, and the cyclic AMP-dependent protein kinase PKA by the regulatory proteins of adeno-associated virus type 2. Mol Cell Biol 18, 59215929.
Di Pasquale, G. & Stacey, S. N. (1998). Adeno-associated virus Rep78 protein interacts with protein kinase A and its homolog PRKX and inhibits CREB-dependent transcriptional activation. J Virol 72, 79167925.
Dyall, J. & Berns, K. I. (1998). Site-specific integration of adeno-associated virus into an episome with the target locus via a deletion-substitution mechanism. J Virol 72, 61956198.
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, 1003910043.
Hermanns, J., Schulze, A., Jansen-Dürr, P., Kleinschmidt, J. A., Schmidt, R. & zur Hausen, H. (1997). Infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins. J Virol 71, 60206027.[Abstract]
Hermonat, P. L. (1991). Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product. Cancer Res 51, 33733377.[Abstract]
Hermonat, P. L. (1994). Down-regulation of the human c-fos and c-myc proto-oncogene promoters by adeno-associated virus Rep78. Cancer Lett 81, 129136.[Medline]
Hölscher, C., Hörer, M., Kleinschmidt, J. A., Zentgraf, H., Bürkle, A. & Heilbronn, R. (1994). Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants. J Virol 68, 71697177.[Abstract]
Im, D.-S. & Muzyczka, N. (1989). Factors that bind to adeno-associated virus terminal repeats. J Virol 63, 30953104.[Medline]
Im, D.-S. & Muzyczka, N. (1990). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61, 447457.[Medline]
Kearns, W. G., Afione, S. A., Fulmer, S. B., Pang, M. C., Erikson, D., Egan, M., Landrum, M. J., Flotte, T. R. & Cutting, G. R. (1996). Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther 3, 748755.[Medline]
Kogure, K., Urabe, M., Mizukami, H., Kume, A., Sato, Y., Monahan, J. & Ozawa, K. (2001). Targeted integration of foreign DNA into a defined locus on chromosome 19 in K562 cells using AAV-derived components. Int J Hematol 73, 469475.[Medline]
Kotin, R. M. (1994). Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther 5, 793801.[Medline]
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, 22112215.[Abstract]
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, 50715078.[Abstract]
Labow, M. A., Graf, L. H., Jr & Berns, K. I. (1987). Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol 7, 13201325.[Medline]
Lamartina, S., Roscilli, G., Rinaudo, D., Delmastro, P. & Toniatti, C. (1998). Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle. J Virol 72, 76537658.
Linden, R. M., Ward, P., Giraud, C., Winocour, E. & Berns, K. I. (1996). Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 93, 1128811294.
McCarty, D. M., Christensen, M. & Muzyczka, N. (1991). Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein. J Virol 65, 29362945.[Medline]
McCarty, D. M., Pereira, D. J., Zolotukhin, I., Zhou, X., Ryan, J. H. & Muzyczka, N. (1994). Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol 68, 49884997.[Abstract]
Muzyczka, N. (1992). Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 158, 97129.[Medline]
Ni, T. H., Zhou, X., McCarty, D. M., Zolotukhin, I. & Muzyczka, N. (1994). In vitro replication of adeno-associated virus DNA. J Virol 68, 11281138.[Abstract]
Ni, T. H., McDonald, W. F., Zolotukhin, I., Melendy, T., Waga, S., Stillman, B. & Muzyczka, N. (1998). Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection. J Virol 72, 27772787.
Oelze, I., Rittner, K. & Sczakiel, G. (1994). Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions. J Virol 68, 12291233.[Abstract]
Pieroni, L., Fipaldini, C., Monciotti, A. & 7 other authors (1998). Targeted integration of adeno-associated virus-derived plasmids in transfected human cells. Virology 249, 249259.[CrossRef][Medline]
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, 39413950.[Abstract]
Shelling, A. N. & Smith, M. G. (1994). Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene. Gene Ther 1, 165169.[Medline]
Surosky, R. T., Urabe, M., Godwin, S. G., McQuiston, S. A., Kurtzman, G. J., Ozawa, K. & Natsoulis, G. (1997). Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol 71, 79517959.[Abstract]
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, 391401.[CrossRef][Medline]
Weger, S., Wistuba, A., Grimm, D. & Kleinschmidt, J. A. (1997). Control of adeno-associated virus type 2 cap gene expression: relative influence of helper virus, terminal repeats, and Rep proteins. J Virol 71, 84378447.[Abstract]
Weitzman, M. D., Kyöstio, S. R., 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, 58085812.[Abstract]
Wollscheid, V., Frey, M., Zentgraf, H. & Sczakiel, G. (1997). Purification and characterization of an active form of the p78Rep protein of adeno-associated virus type 2 expressed in Escherichia coli. Protein Expr Purif 11, 241249.[CrossRef][Medline]
Yang, Q., Kadam, A. & Trempe, J. P. (1992). Mutational analysis of the adeno-associated virus rep gene. J Virol 66, 60586069.[Abstract]
Received 3 March 2003;
accepted 1 April 2003.