Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Sep;71(9):6996–7004. doi: 10.1128/jvi.71.9.6996-7004.1997

Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

S L Walker 1, R S Wonderling 1, R A Owens 1
PMCID: PMC191985  PMID: 9261429

Abstract

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Afanasiev B. N., Galyov E. E., Buchatsky L. P., Kozlov Y. V. Nucleotide sequence and genomic organization of Aedes densonucleosis virus. Virology. 1991 Nov;185(1):323–336. doi: 10.1016/0042-6822(91)90780-f. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Astell C. R., Gardiner E. M., Tattersall P. DNA sequence of the lymphotropic variant of minute virus of mice, MVM(i), and comparison with the DNA sequence of the fibrotropic prototype strain. J Virol. 1986 Feb;57(2):656–669. doi: 10.1128/jvi.57.2.656-669.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bloom M. E., Alexandersen S., Perryman S., Lechner D., Wolfinbarger J. B. Nucleotide sequence and genomic organization of Aleutian mink disease parvovirus (ADV): sequence comparisons between a nonpathogenic and a pathogenic strain of ADV. J Virol. 1988 Aug;62(8):2903–2915. doi: 10.1128/jvi.62.8.2903-2915.1988. [DOI] [PMC free article] [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 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]
  9. Chiorini J. A., Weitzman M. D., Owens R. A., Urcelay E., Safer B., Kotin R. M. Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli. J Virol. 1994 Feb;68(2):797–804. doi: 10.1128/jvi.68.2.797-804.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Giraud C., Winocour E., Berns K. I. Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10039–10043. doi: 10.1073/pnas.91.21.10039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Wolf Y. I. A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses. FEBS Lett. 1990 Mar 12;262(1):145–148. doi: 10.1016/0014-5793(90)80175-i. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hermonat P. L. Down-regulation of the human c-fos and c-myc proto-oncogene promoters by adeno-associated virus Rep78. Cancer Lett. 1994 Jun 30;81(2):129–136. doi: 10.1016/0304-3835(94)90193-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  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. Jindal H. K., Yong C. B., Wilson G. M., Tam P., Astell C. R. Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions. J Biol Chem. 1994 Feb 4;269(5):3283–3289. [PubMed] [Google Scholar]
  20. Koonin E. V. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res. 1993 Jun 11;21(11):2541–2547. doi: 10.1093/nar/21.11.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kotin R. M., Linden R. M., Berns K. I. Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 1992 Dec;11(13):5071–5078. doi: 10.1002/j.1460-2075.1992.tb05614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kotin R. M., Menninger J. C., Ward D. C., Berns K. I. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. doi: 10.1016/0888-7543(91)90470-y. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kyöstiö S. R., Owens R. A. Identification of mutant adeno-associated virus Rep proteins which are dominant-negative for DNA helicase activity. Biochem Biophys Res Commun. 1996 Mar 18;220(2):294–299. doi: 10.1006/bbrc.1996.0399. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kyöstiö S. R., Wonderling R. S., Owens R. A. Negative regulation of the adeno-associated virus (AAV) P5 promoter involves both the P5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein. J Virol. 1995 Nov;69(11):6787–6796. doi: 10.1128/jvi.69.11.6787-6796.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Linden R. M., Winocour E., Berns K. I. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966–7972. doi: 10.1073/pnas.93.15.7966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lohman T. M. Helicase-catalyzed DNA unwinding. J Biol Chem. 1993 Feb 5;268(4):2269–2272. [PubMed] [Google Scholar]
  30. McCarty D. M., Ni T. H., Muzyczka N. Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro. J Virol. 1992 Jul;66(7):4050–4057. doi: 10.1128/jvi.66.7.4050-4057.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Owens R. A., Trempe J. P., Chejanovsky N., Carter B. J. Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin. Virology. 1991 Sep;184(1):14–22. doi: 10.1016/0042-6822(91)90817-u. [DOI] [PubMed] [Google Scholar]
  34. Owens R. A., Weitzman M. D., Kyöstiö S. R., Carter B. J. Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins. J Virol. 1993 Feb;67(2):997–1005. doi: 10.1128/jvi.67.2.997-1005.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Samulski R. J., Srivastava A., Berns K. I., Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983 May;33(1):135–143. doi: 10.1016/0092-8674(83)90342-2. [DOI] [PubMed] [Google Scholar]
  37. Saraste M., Sibbald P. R., Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. doi: 10.1016/0968-0004(90)90281-f. [DOI] [PubMed] [Google Scholar]
  38. Senapathy P., Tratschin J. D., Carter B. J. Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions. J Mol Biol. 1984 Oct 15;179(1):1–20. doi: 10.1016/0022-2836(84)90303-6. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. 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]
  44. Thomson B. J., Efstathiou S., Honess R. W. Acquisition of the human adeno-associated virus type-2 rep gene by human herpesvirus type-6. Nature. 1991 May 2;351(6321):78–80. doi: 10.1038/351078a0. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Urcelay E., Ward P., Wiener S. M., Safer B., Kotin R. M. Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein. J Virol. 1995 Apr;69(4):2038–2046. doi: 10.1128/jvi.69.4.2038-2046.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Walker S. L., Wonderling R. S., Owens R. A. Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity. J Virol. 1997 Apr;71(4):2722–2730. doi: 10.1128/jvi.71.4.2722-2730.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Warrener P., Tamura J. K., Collett M. S. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J Virol. 1993 Feb;67(2):989–996. doi: 10.1128/jvi.67.2.989-996.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weitzman M. D., Kyöstiö S. R., Kotin R. M., Owens R. A. 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. 1994 Jun 21;91(13):5808–5812. doi: 10.1073/pnas.91.13.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wonderling R. S., Kyöstiö S. R., Owens R. A. A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. J Virol. 1995 Jun;69(6):3542–3548. doi: 10.1128/jvi.69.6.3542-3548.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wonderling R. S., Owens R. A. Binding sites for adeno-associated virus Rep proteins within the human genome. J Virol. 1997 Mar;71(3):2528–2534. doi: 10.1128/jvi.71.3.2528-2534.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wonderling R. S., Owens R. A. The Rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene. J Virol. 1996 Jul;70(7):4783–4786. doi: 10.1128/jvi.70.7.4783-4786.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yu X., Egelman E. H. Direct visualization of dynamics and co-operative conformational changes within RecA filaments that appear to be associated with the hydrolysis of adenosine 5'-O-(3-thiotriphosphate). J Mol Biol. 1992 May 5;225(1):193–216. doi: 10.1016/0022-2836(92)91036-o. [DOI] [PubMed] [Google Scholar]
  55. Zádori Z., Stefancsik R., Rauch T., Kisary J. Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2. Virology. 1995 Oct 1;212(2):562–573. doi: 10.1006/viro.1995.1514. [DOI] [PubMed] [Google Scholar]

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

RESOURCES