Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1990 Apr;64(4):1764–1770. doi: 10.1128/jvi.64.4.1764-1770.1990

Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication.

N Chejanovsky 1, B J Carter 1
PMCID: PMC249314  PMID: 2157057

Abstract

Adeno-associated virus (AAV) contains a multifunctional nonstructural gene, rep, which is required for AAV DNA replication and has pleiotropic effects on positive and negative regulation of gene expression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conserved region is the consensus sequence for a purine nucleotide binding site. We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced the mutant Rep proteins, indicating that these proteins are autoregulated. Furthermore, the mutant gene was unable to replicate but was able to inhibit in trans wild-type AAV DNA replication. Thus, pNTC23 represents a dominant negative mutant of AAV. These results suggest that rep has separate functional domains important for DNA replication.

Full text

PDF
1764

Images in this article

1766Image
on p.1766
1766Image
on p.1766
1767Image
on p.1767
1767Image
on p.1767
1768Image
on p.1768
1769Image
on p.1769

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. Astell C. R., Mol C. D., Anderson W. F. Structural and functional homology of parvovirus and papovavirus polypeptides. J Gen Virol. 1987 Mar;68(Pt 3):885–893. doi: 10.1099/0022-1317-68-3-885. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bradley M. K., Smith T. F., Lathrop R. H., Livingston D. M., Webster T. A. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4026–4030. doi: 10.1073/pnas.84.12.4026. [DOI] [PMC free article] [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. 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]
  7. Chen K. C., Shull B. C., Moses E. A., Lederman M., Stout E. R., Bates R. C. Complete nucleotide sequence and genome organization of bovine parvovirus. J Virol. 1986 Dec;60(3):1085–1097. doi: 10.1128/jvi.60.3.1085-1097.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
  9. Clark R., Peden K., Pipas J. M., Nathans D., Tjian R. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol Cell Biol. 1983 Feb;3(2):220–228. doi: 10.1128/mcb.3.2.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clertant P., Cuzin F. Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses. J Biol Chem. 1982 Jun 10;257(11):6300–6305. [PubMed] [Google Scholar]
  11. Clertant P., Seif I. A common function for polyoma virus large-T and papillomavirus E1 proteins? Nature. 1984 Sep 20;311(5983):276–279. doi: 10.1038/311276a0. [DOI] [PubMed] [Google Scholar]
  12. Cole C. N., Tornow J., Clark R., Tjian R. Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A. J Virol. 1986 Feb;57(2):539–546. doi: 10.1128/jvi.57.2.539-546.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Denhez F., Heimann B., d'Auriol L., Graf T., Coquillaud M., Coll J., Galibert F., Moelling K., Stehelin D., Ghysdael J. Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus. EMBO J. 1988 Feb;7(2):541–546. doi: 10.1002/j.1460-2075.1988.tb02843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  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. Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [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. 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]
  19. Labow M. A., Hermonat P. L., Berns K. I. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. doi: 10.1128/jvi.60.1.251-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Mendelson E., Trempe J. P., Carter B. J. Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. J Virol. 1986 Dec;60(3):823–832. doi: 10.1128/jvi.60.3.823-832.1986. [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., 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]
  24. Shade R. O., Blundell M. C., Cotmore S. F., Tattersall P., Astell C. R. Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. J Virol. 1986 Jun;58(3):921–936. doi: 10.1128/jvi.58.3.921-936.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Straus S. E., Sebring E. D., Rose J. A. Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci U S A. 1976 Mar;73(3):742–746. doi: 10.1073/pnas.73.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Trempe J. P., Carter B. J. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation. J Virol. 1988 Jan;62(1):68–74. doi: 10.1128/jvi.62.1.68-74.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trempe J. P., Mendelson E., Carter B. J. Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli. Virology. 1987 Nov;161(1):18–28. doi: 10.1016/0042-6822(87)90166-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES