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. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065

Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration.

T Fujiwara 1, R Craigie 1
PMCID: PMC287065  PMID: 2524066

Abstract

After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome. Integration of this DNA copy into the host genome is a necessary step for efficient viral replication. We have developed a cell-free system for integration of exogenous mini-retroviral DNA. The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA. The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles. Phage lambda DNA serves as the target for integration. Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli. Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture. The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence. In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA.

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Selected References

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

  1. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  2. Cobrinik D., Katz R., Terry R., Skalka A. M., Leis J. Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth. J Virol. 1987 Jun;61(6):1999–2008. doi: 10.1128/jvi.61.6.1999-2008.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Colicelli J., Goff S. P. Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site. Cell. 1985 Sep;42(2):573–580. doi: 10.1016/0092-8674(85)90114-x. [DOI] [PubMed] [Google Scholar]
  4. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  5. Craigie R., Mizuuchi K. Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate. Cell. 1985 Jul;41(3):867–876. doi: 10.1016/s0092-8674(85)80067-2. [DOI] [PubMed] [Google Scholar]
  6. Duyk G., Longiaru M., Cobrinik D., Kowal R., deHaseth P., Skalka A. M., Leis J. Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage. J Virol. 1985 Nov;56(2):589–599. doi: 10.1128/jvi.56.2.589-599.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fan H., Baltimore D. RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA. J Mol Biol. 1973 Oct 15;80(1):93–117. doi: 10.1016/0022-2836(73)90235-0. [DOI] [PubMed] [Google Scholar]
  8. Fujiwara T., Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. doi: 10.1016/0092-8674(88)90071-2. [DOI] [PubMed] [Google Scholar]
  9. Grandgenett D. P., Vora A. C., Swanstrom R., Olsen J. C. Nuclease mechanism of the avian retrovirus pp32 endonuclease. J Virol. 1986 Jun;58(3):970–974. doi: 10.1128/jvi.58.3.970-974.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Panganiban A. T., Temin H. M. Circles with two tandem LTRs are precursors to integrated retrovirus DNA. Cell. 1984 Mar;36(3):673–679. doi: 10.1016/0092-8674(84)90347-7. [DOI] [PubMed] [Google Scholar]
  11. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  12. Shoemaker C., Goff S., Gilboa E., Paskind M., Mitra S. W., Baltimore D. Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3932–3936. doi: 10.1073/pnas.77.7.3932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tanese N., Roth M. J., Goff S. P. Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli. J Virol. 1986 Aug;59(2):328–340. doi: 10.1128/jvi.59.2.328-340.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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