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. 1994 Sep;68(9):5690–5697. doi: 10.1128/jvi.68.9.5690-5697.1994

Detection and characterization of a functional complex of human immunodeficiency virus type 1 integrase and its DNA substrate by UV cross-linking.

T Yoshinaga 1, Y Kimura-Ohtani 1, T Fujiwara 1
PMCID: PMC236971  PMID: 8057450

Abstract

To analyze the association of the human immunodeficiency virus type 1 integrase (IN) protein with DNA substrates, we applied a shortwave UV cross-linking method to the in vitro integration system. Three photoadduct bands were detected on sodium dodecyl sulfate-polyacrylamide gels. The appearances of these photoadducts were examined with various substrates and under several incubation conditions. Our data suggest that one of these photoadducts is derived from the sequence- and strand-specific bound state of the early phase of the integration reaction before the 3' processing reaction. We also show that most of the photoadduct complexes are competent for integration even after UV irradiation.

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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. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bushman F. D., Craigie R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1339–1343. doi: 10.1073/pnas.88.4.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bushman F. D., Craigie R. Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro. J Virol. 1990 Nov;64(11):5645–5648. doi: 10.1128/jvi.64.11.5645-5648.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bushman F. D., Fujiwara T., Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. doi: 10.1126/science.2171144. [DOI] [PubMed] [Google Scholar]
  5. Chow S. A., Vincent K. A., Ellison V., Brown P. O. Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science. 1992 Feb 7;255(5045):723–726. doi: 10.1126/science.1738845. [DOI] [PubMed] [Google Scholar]
  6. Craigie R., Fujiwara T., Bushman F. The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell. 1990 Aug 24;62(4):829–837. doi: 10.1016/0092-8674(90)90126-y. [DOI] [PubMed] [Google Scholar]
  7. Engelman A., Bushman F. D., Craigie R. Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex. EMBO J. 1993 Aug;12(8):3269–3275. doi: 10.1002/j.1460-2075.1993.tb05996.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engelman A., Mizuuchi K., Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell. 1991 Dec 20;67(6):1211–1221. doi: 10.1016/0092-8674(91)90297-c. [DOI] [PubMed] [Google Scholar]
  9. Fesen M. R., Kohn K. W., Leteurtre F., Pommier Y. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2399–2403. doi: 10.1073/pnas.90.6.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujiwara T., Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Goff S. P. Genetics of retroviral integration. Annu Rev Genet. 1992;26:527–544. doi: 10.1146/annurev.ge.26.120192.002523. [DOI] [PubMed] [Google Scholar]
  13. Hazuda D. J., Wolfe A. L., Hastings J. C., Robbins H. L., Graham P. L., LaFemina R. L., Emini E. A. Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase. J Biol Chem. 1994 Feb 11;269(6):3999–4004. [PubMed] [Google Scholar]
  14. Hizi A., Hughes S. H. Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli. Virology. 1988 Dec;167(2):634–638. [PubMed] [Google Scholar]
  15. Ishimoto L. K., Halperin M., Champoux J. J. Moloney murine leukemia virus IN protein from disrupted virions binds and specifically cleaves its target sequence in vitro. Virology. 1991 Feb;180(2):527–534. doi: 10.1016/0042-6822(91)90066-k. [DOI] [PubMed] [Google Scholar]
  16. Jonsson C. B., Roth M. J. Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration. J Virol. 1993 Sep;67(9):5562–5571. doi: 10.1128/jvi.67.9.5562-5571.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katz R. A., Merkel G., Kulkosky J., Leis J., Skalka A. M. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. doi: 10.1016/0092-8674(90)90290-u. [DOI] [PubMed] [Google Scholar]
  18. Katzman M., Katz R. A., Skalka A. M., Leis J. The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration. J Virol. 1989 Dec;63(12):5319–5327. doi: 10.1128/jvi.63.12.5319-5327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krogstad P. A., Champoux J. J. Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein. J Virol. 1990 Jun;64(6):2796–2801. doi: 10.1128/jvi.64.6.2796-2801.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LaFemina R. L., Callahan P. L., Cordingley M. G. Substrate specificity of recombinant human immunodeficiency virus integrase protein. J Virol. 1991 Oct;65(10):5624–5630. doi: 10.1128/jvi.65.10.5624-5630.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luk K. C., Gilmore T. D., Panganiban A. T. The spleen necrosis virus int gene product expressed in Escherichia coli has DNA binding activity and mediates att and U5-specific DNA multimer formation in vitro. Virology. 1987 Mar;157(1):127–136. doi: 10.1016/0042-6822(87)90321-7. [DOI] [PubMed] [Google Scholar]
  23. Misra T. K., Grandgenett D. P., Parsons J. T. Avian retrovirus pp32 DNA-binding protein. I. Recognition of specific sequences on retrovirus DNA terminal repeats. J Virol. 1982 Oct;44(1):330–343. doi: 10.1128/jvi.44.1.330-343.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mumm S. R., Grandgenett D. P. Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis. J Virol. 1991 Mar;65(3):1160–1167. doi: 10.1128/jvi.65.3.1160-1167.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murphy J. E., Goff S. P. A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo. J Virol. 1992 Aug;66(8):5092–5095. doi: 10.1128/jvi.66.8.5092-5095.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  27. Roth M. J., Schwartzberg P., Tanese N., Goff S. P. Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting. J Virol. 1990 Oct;64(10):4709–4717. doi: 10.1128/jvi.64.10.4709-4717.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roth M. J., Tanese N., Goff S. P. Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein. J Mol Biol. 1988 Sep 5;203(1):131–139. doi: 10.1016/0022-2836(88)90097-6. [DOI] [PubMed] [Google Scholar]
  29. Schauer M., Billich A. The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding. Biochem Biophys Res Commun. 1992 Jun 30;185(3):874–880. doi: 10.1016/0006-291x(92)91708-x. [DOI] [PubMed] [Google Scholar]
  30. Sherman P. A., Dickson M. L., Fyfe J. A. Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions. J Virol. 1992 Jun;66(6):3593–3601. doi: 10.1128/jvi.66.6.3593-3601.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sherman P. A., Fyfe J. A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119–5123. doi: 10.1073/pnas.87.13.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Terry R., Soltis D. A., Katzman M., Cobrinik D., Leis J., Skalka A. M. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vink C., van Gent D. C., Elgersma Y., Plasterk R. H. Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J Virol. 1991 Sep;65(9):4636–4644. doi: 10.1128/jvi.65.9.4636-4644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vora A. C., Fitzgerald M. L., Grandgenett D. P. Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein. J Virol. 1990 Nov;64(11):5656–5659. doi: 10.1128/jvi.64.11.5656-5659.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
  36. Woerner A. M., Klutch M., Levin J. G., Marcus-Sekura C. J. Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein. AIDS Res Hum Retroviruses. 1992 Feb;8(2):297–304. doi: 10.1089/aid.1992.8.297. [DOI] [PubMed] [Google Scholar]
  37. van Gent D. C., Elgersma Y., Bolk M. W., Vink C., Plasterk R. H. DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2. Nucleic Acids Res. 1991 Jul 25;19(14):3821–3827. doi: 10.1093/nar/19.14.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Gent D. C., Vink C., Groeneger A. A., Plasterk R. H. Complementation between HIV integrase proteins mutated in different domains. EMBO J. 1993 Aug;12(8):3261–3267. doi: 10.1002/j.1460-2075.1993.tb05995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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