Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):6880–6885. doi: 10.1128/jvi.69.11.6880-6885.1995

High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant.

C Hölscher 1, J A Kleinschmidt 1, A Bürkle 1
PMCID: PMC189603  PMID: 7474103

Abstract

Adeno-associated virus (AAV) codes for four closely related nonstructural proteins (Rep) required for AAV DNA replication and gene regulation. In vitro studies have revealed that either Rep78 or Rep68 alone is sufficient for AAV DNA replication. Rep52 and Rep40 are not required for DNA replication but have been reported to enhance the efficiency of accumulation of single-stranded progeny DNA. Previous studies on rep-expressing cell lines had indicated that only a subset of the four Rep proteins are required for the production of infectious AAV. We therefore set out to determine the minimal set of Rep proteins sufficient for the generation of infectious AAV. Transient cotransfections in HeLa cells of constructs for high-level expression of individual Rep proteins with a rep-negative AAV genome revealed that either Rep78 or Rep68 alone could complement for a full replication cycle yielding infectious virus. This result was confirmed by transfection studies in the cell line HeM2, which selectively expresses Rep78 at rather low levels under the control of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat (C. Hölscher, M. Hörer, J. A. Kleinschmidt, H. Zentgraf, A. Bürkle, and R. Heilbronn, J. Virol. 68:7169-7177, 1994). Increasing the level of Rep78 expression by transfection of a glucocorticoid receptor expression construct resulted in a higher level of DNA replication of a cotransfected rep-negative AAV genome and in the production of infectious rep-negative AAV particles. We further report on the generation of a new rep-expressing cell line, HeCM1, which was obtained by stable supertransfection of a construct for constitutive Rep40 expression into HeM1 cells (Hölscher et al., J. Virol. 68:7169-7177). Transfection of rather large amounts of rep-negative AAV DNA led to detectable virus production in HeCM1 cells even in the absence of the cotransfected glucocorticoid receptor expression construct, but higher yields were obtained after increasing the Rep78 level by coexpression of the glucocorticoid receptor. These data demonstrate that all Rep functions required for the productive replication of AAV in HeLa cells are contained within both Rep78 and Rep68.

Full Text

The Full Text of this article is available as a PDF (542.3 KB).

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Cater J. E., Pintel D. J. The small non-structural protein NS2 of the autonomous parvovirus minute virus of mice is required for virus growth in murine cells. J Gen Virol. 1992 Jul;73(Pt 7):1839–1843. doi: 10.1099/0022-1317-73-7-1839. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Fisher R. E., Mayor H. D. The evolution of defective and autonomous parvoviruses. J Theor Biol. 1991 Apr 21;149(4):429–439. doi: 10.1016/s0022-5193(05)80091-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Hölscher C., Hörer M., Kleinschmidt J. A., Zentgraf H., Bürkle A., Heilbronn R. Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants. J Virol. 1994 Nov;68(11):7169–7177. doi: 10.1128/jvi.68.11.7169-7177.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Ko M. S., Takano T. A highly inducible system of gene expression by positive feedback production of glucocorticoid receptors. DNA. 1989 Mar;8(2):127–133. doi: 10.1089/dna.1.1989.8.127. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kumar V., Green S., Stack G., Berry M., Jin J. R., Chambon P. Functional domains of the human estrogen receptor. Cell. 1987 Dec 24;51(6):941–951. doi: 10.1016/0092-8674(87)90581-2. [DOI] [PubMed] [Google Scholar]
  18. McCarty D. M., Pereira D. J., Zolotukhin I., Zhou X., Ryan J. H., Muzyczka N. Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol. 1994 Aug;68(8):4988–4997. doi: 10.1128/jvi.68.8.4988-4997.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McCarty D. M., Ryan J. H., Zolotukhin S., Zhou X., Muzyczka N. Interaction of the adeno-associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat. J Virol. 1994 Aug;68(8):4998–5006. doi: 10.1128/jvi.68.8.4998-5006.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Naeger L. K., Cater J., Pintel D. J. The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner. J Virol. 1990 Dec;64(12):6166–6175. doi: 10.1128/jvi.64.12.6166-6175.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Ward P., Urcelay E., Kotin R., Safer B., Berns K. I. Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein. J Virol. 1994 Sep;68(9):6029–6037. doi: 10.1128/jvi.68.9.6029-6037.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wistuba A., Weger S., Kern A., Kleinschmidt J. A. Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J Virol. 1995 Sep;69(9):5311–5319. doi: 10.1128/jvi.69.9.5311-5319.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  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]

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

RESOURCES