Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Nov;68(11):7169–7177. doi: 10.1128/jvi.68.11.7169-7177.1994

Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants.

C Hölscher 1, M Hörer 1, J A Kleinschmidt 1, H Zentgraf 1, A Bürkle 1, R Heilbronn 1
PMCID: PMC237156  PMID: 7933099

Abstract

The adeno-associated virus (AAV) rep gene codes for a family of nonstructural proteins which are required for AAV gene regulation and DNA replication. In addition, rep has been implicated in a variety of activities outside the AAV life cycle which have been difficult to study, since attempts to achieve separate and constitutive expression of rep in stable cell lines have failed so far. Here we report the generation of two cell lines which inducibly express Rep78 under the control of the glucocorticoid-responsive mouse mammary tumor virus promoter. In addition, one of the cell lines constitutively expresses relatively high levels of Rep52. Both cell lines showed similar plating efficiencies with and without induction of Rep78 expression, which rules out cytotoxic effects of Rep78. The cell lines efficiently support DNA replication of a rep-negative AAV genome and initiate the formation of AAV particles. However, despite the correct sizes and stoichiometry of the three capsid proteins, the AAV particles were noninfectious. This was found to be due to a defect in the accumulation of single-stranded AAV DNA. Transient transfection of single expression constructs for constitutive, high-level expression of individual Rep proteins (either Rep78, Rep68, Rep52, or Rep40) complemented this defect. Infectious rep-negative AAV progeny was produced at varying efficiencies depending on the rep expression construct used. These data show that functional expression of full-length Rep in recombinant cell lines is possible and that the state of Rep expression is critical for the infectivity of AAV progeny produced.

Full text

PDF
7172

Images in this article

7170Image
on p.7170
7171Image
on p.7171
7172Image
on p.7172
7173Image
on p.7173
7174Image
on p.7174
7175Image
on p.7175

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beaton A., Palumbo P., Berns K. I. 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. doi: 10.1128/jvi.63.10.4450-4454.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berns K. I. Parvovirus replication. Microbiol Rev. 1990 Sep;54(3):316–329. doi: 10.1128/mr.54.3.316-329.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buller R. M., Rose J. A. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J Virol. 1978 Jan;25(1):331–338. doi: 10.1128/jvi.25.1.331-338.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caillet-Fauquet P., Perros M., Brandenburger A., Spegelaere P., Rommelaere J. Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins. EMBO J. 1990 Sep;9(9):2989–2995. doi: 10.1002/j.1460-2075.1990.tb07491.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter B. J., Marcus-Sekura C. J., Laughlin C. A., Ketner G. Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication. Virology. 1983 Apr 30;126(2):505–516. doi: 10.1016/s0042-6822(83)80008-7. [DOI] [PubMed] [Google Scholar]
  6. Chejanovsky N., Carter B. J. Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication. Virology. 1989 Nov;173(1):120–128. doi: 10.1016/0042-6822(89)90227-4. [DOI] [PubMed] [Google Scholar]
  7. Chejanovsky N., Carter B. J. Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor. Virology. 1989 Jul;171(1):239–247. doi: 10.1016/0042-6822(89)90531-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Fasel N., Pearson K., Buetti E., Diggelmann H. The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription. EMBO J. 1982;1(1):3–7. doi: 10.1002/j.1460-2075.1982.tb01115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hauswirth W. W., Berns K. I. Adeno-associated virus DNA replication: nonunit-length molecules. Virology. 1979 Feb;93(1):57–68. doi: 10.1016/0042-6822(79)90275-7. [DOI] [PubMed] [Google Scholar]
  11. Hauswirth W. W., Berns K. I. Origin and termination of adeno-associated virus DNA replication. Virology. 1977 May 15;78(2):488–499. doi: 10.1016/0042-6822(77)90125-8. [DOI] [PubMed] [Google Scholar]
  12. Heilbronn R., Bürkle A., Stephan S., zur Hausen H. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification. J Virol. 1990 Jun;64(6):3012–3018. doi: 10.1128/jvi.64.6.3012-3018.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heilbronn R., zur Hausen H. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome. J Virol. 1989 Sep;63(9):3683–3692. doi: 10.1128/jvi.63.9.3683-3692.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hermonat P. L. Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product. Cancer Res. 1991 Jul 1;51(13):3373–3377. [PubMed] [Google Scholar]
  15. Hermonat P. L., Labow M. A., Wright R., Berns K. I., Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 1984 Aug;51(2):329–339. doi: 10.1128/jvi.51.2.329-339.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hermonat P. L. The adeno-associated virus Rep78 gene inhibits cellular transformation induced by bovine papillomavirus. Virology. 1989 Sep;172(1):253–261. doi: 10.1016/0042-6822(89)90127-x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Janik J. E., Huston M. M., Cho K., Rose J. A. Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA. Virology. 1989 Feb;168(2):320–329. doi: 10.1016/0042-6822(89)90272-9. [DOI] [PubMed] [Google Scholar]
  20. Khleif S. N., Myers T., Carter B. J., Trempe J. P. Inhibition of cellular transformation by the adeno-associated virus rep gene. Virology. 1991 Apr;181(2):738–741. doi: 10.1016/0042-6822(91)90909-u. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kyöstiö S. R., Owens R. A., Weitzman M. D., Antoni B. A., Chejanovsky N., Carter B. J. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels. J Virol. 1994 May;68(5):2947–2957. doi: 10.1128/jvi.68.5.2947-2957.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Labow M. A., Graf L. H., Jr, Berns K. I. Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol. 1987 Apr;7(4):1320–1325. doi: 10.1128/mcb.7.4.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lusby E., Bohenzky R., Berns K. I. Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome. J Virol. 1981 Mar;37(3):1083–1086. doi: 10.1128/jvi.37.3.1083-1086.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lusby E., Fife K. H., Berns K. I. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol. 1980 May;34(2):402–409. doi: 10.1128/jvi.34.2.402-409.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Myers M. W., Carter B. J. Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA. J Biol Chem. 1981 Jan 25;256(2):567–570. [PubMed] [Google Scholar]
  30. Ni T. H., Zhou X., McCarty D. M., Zolotukhin I., Muzyczka N. In vitro replication of adeno-associated virus DNA. J Virol. 1994 Feb;68(2):1128–1138. doi: 10.1128/jvi.68.2.1128-1138.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Redemann B. E., Mendelson E., Carter B. J. Adeno-associated virus rep protein synthesis during productive infection. J Virol. 1989 Feb;63(2):873–882. doi: 10.1128/jvi.63.2.873-882.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rommelaere J., Cornelis J. J. Antineoplastic activity of parvoviruses. J Virol Methods. 1991 Aug;33(3):233–251. doi: 10.1016/0166-0934(91)90024-t. [DOI] [PubMed] [Google Scholar]
  33. Ruffing M., Zentgraf H., Kleinschmidt J. A. Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells. J Virol. 1992 Dec;66(12):6922–6930. doi: 10.1128/jvi.66.12.6922-6930.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Tratschin J. D., Miller I. L., Carter B. J. 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. doi: 10.1128/jvi.51.3.611-619.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tratschin J. D., Tal J., Carter B. J. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. doi: 10.1128/mcb.6.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Weindler F. W., Heilbronn R. A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J Virol. 1991 May;65(5):2476–2483. doi: 10.1128/jvi.65.5.2476-2483.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yalkinoglu A. O., Heilbronn R., Bürkle A., Schlehofer J. R., zur Hausen H. DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res. 1988 Jun 1;48(11):3123–3129. [PubMed] [Google Scholar]
  44. 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]
  45. Yang Q., Kadam A., Trempe J. P. Mutational analysis of the adeno-associated virus rep gene. J Virol. 1992 Oct;66(10):6058–6069. doi: 10.1128/jvi.66.10.6058-6069.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhao Q., Schoborg R. V., Pintel D. J. Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron. J Virol. 1994 May;68(5):2849–2859. doi: 10.1128/jvi.68.5.2849-2859.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES