Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1987 Oct;61(10):3096–3101. doi: 10.1128/jvi.61.10.3096-3101.1987

A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.

R J Samulski, L S Chang, T Shenk
PMCID: PMC255885  PMID: 3041032

Abstract

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.

Full text

PDF
3096

Images in this article

3098Image
on p.3098
3099Image
on p.3099
3100Image
on p.3100

Selected References

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

  1. Been M. D., Champoux J. J. Cutting of M13mp7 phage DNA and excision of cloned single-stranded sequences by restriction endonucleases. Methods Enzymol. 1983;101:90–98. doi: 10.1016/0076-6879(83)01007-1. [DOI] [PubMed] [Google Scholar]
  2. Berns K. I., Adler S. Separation of two types of adeno-associated virus particles containing complementary polynucleotide chains. J Virol. 1972 Feb;9(2):394–396. doi: 10.1128/jvi.9.2.394-396.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berns K. I., Rose J. A. Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands. J Virol. 1970 Jun;5(6):693–699. doi: 10.1128/jvi.5.6.693-699.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blacklow N. R., Hoggan M. D., Rowe W. P. Immunofluorescent studies of the potentiation of an adenovirus-associated virus by adenovirus 7. J Exp Med. 1967 May 1;125(5):755–765. doi: 10.1084/jem.125.5.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheung A. K., Hoggan M. D., Hauswirth W. W., Berns K. I. Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol. 1980 Feb;33(2):739–748. doi: 10.1128/jvi.33.2.739-748.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dotto G. P., Horiuchi K. Replication of a plasmid containing two origins of bacteriophage. J Mol Biol. 1981 Nov 25;153(1):169–176. doi: 10.1016/0022-2836(81)90532-5. [DOI] [PubMed] [Google Scholar]
  8. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  11. Jones N., Shenk T. Isolation of deletion and substitution mutants of adenovirus type 5. Cell. 1978 Jan;13(1):181–188. doi: 10.1016/0092-8674(78)90148-4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Laughlin C. A., Westphal H., Carter B. J. Spliced adenovirus-associated virus RNA. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5567–5571. doi: 10.1073/pnas.76.11.5567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lusby E. W., Berns K. I. Mapping of the 5' termini of two adeno-associated virus 2 RNAs in the left half of the genome. J Virol. 1982 Feb;41(2):518–526. doi: 10.1128/jvi.41.2.518-526.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Marcus C. J., Laughlin C. A., Carter B. J. Adeno-associated virus RNA transcription in vivo. Eur J Biochem. 1981 Dec;121(1):147–154. doi: 10.1111/j.1432-1033.1981.tb06443.x. [DOI] [PubMed] [Google Scholar]
  16. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  17. 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]
  18. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  19. Rose J. A., Berns K. I., Hoggan M. D., Koczot F. J. Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci U S A. 1969 Nov;64(3):863–869. doi: 10.1073/pnas.64.3.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Samulski R. J., Berns K. I., Tan M., Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. doi: 10.1073/pnas.79.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Yakobson B., Koch T., Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol. 1987 Apr;61(4):972–981. doi: 10.1128/jvi.61.4.972-981.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES