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. 1994 Sep;68(9):5911–5917. doi: 10.1128/jvi.68.9.5911-5917.1994

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

A Engelman 1, A B Hickman 1, R Craigie 1
PMCID: PMC236996  PMID: 8057470

Abstract

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.

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Selected References

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

  1. 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]
  2. Bushman F. D., Craigie R. Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3458–3462. doi: 10.1073/pnas.89.8.3458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bushman F. D., Engelman A., Palmer I., Wingfield P., Craigie R. Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3428–3432. doi: 10.1073/pnas.90.8.3428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Drelich M., Haenggi M., Mous J. Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate. J Virol. 1993 Aug;67(8):5041–5044. doi: 10.1128/jvi.67.8.5041-5044.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drelich M., Wilhelm R., Mous J. Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology. 1992 Jun;188(2):459–468. doi: 10.1016/0042-6822(92)90499-f. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Engelman A., Craigie R. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J Virol. 1992 Nov;66(11):6361–6369. doi: 10.1128/jvi.66.11.6361-6369.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Fayet O., Ramond P., Polard P., Prère M. F., Chandler M. Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol. 1990 Oct;4(10):1771–1777. doi: 10.1111/j.1365-2958.1990.tb00555.x. [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. Grandgenett D. P., Vora A. C., Schiff R. D. A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity. Virology. 1978 Aug;89(1):119–132. doi: 10.1016/0042-6822(78)90046-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Kalpana G. V., Goff S. P. Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10593–10597. doi: 10.1073/pnas.90.22.10593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Knaus R. J., Hippenmeyer P. J., Misra T. K., Grandgenett D. P., Müller U. R., Fitch W. M. Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA. Biochemistry. 1984 Jan 17;23(2):350–359. doi: 10.1021/bi00297a026. [DOI] [PubMed] [Google Scholar]
  18. Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. LaFemina R. L., Schneider C. L., Robbins H. L., Callahan P. L., LeGrow K., Roth E., Schleif W. A., Emini E. A. Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. J Virol. 1992 Dec;66(12):7414–7419. doi: 10.1128/jvi.66.12.7414-7419.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leavitt A. D., Rose R. B., Varmus H. E. Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae. J Virol. 1992 Apr;66(4):2359–2368. doi: 10.1128/jvi.66.4.2359-2368.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leavitt A. D., Shiue L., Varmus H. E. Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J Biol Chem. 1993 Jan 25;268(3):2113–2119. [PubMed] [Google Scholar]
  23. Mazumder A., Engelman A., Craigie R., Fesen M., Pommier Y. Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase. Nucleic Acids Res. 1994 Mar 25;22(6):1037–1043. doi: 10.1093/nar/22.6.1037. [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. 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]
  26. Rowland S. J., Dyke K. G. Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol. 1990 Jun;4(6):961–975. doi: 10.1111/j.1365-2958.1990.tb00669.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Vink C., Oude Groeneger A. M., Plasterk R. H. Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein. Nucleic Acids Res. 1993 Mar 25;21(6):1419–1425. doi: 10.1093/nar/21.6.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vink C., Plasterk R. H. The human immunodeficiency virus integrase protein. Trends Genet. 1993 Dec;9(12):433–438. doi: 10.1016/0168-9525(93)90107-s. [DOI] [PubMed] [Google Scholar]
  32. Vink C., Yeheskiely E., van der Marel G. A., van Boom J. H., Plasterk R. H. Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNA termini mediated by the viral integrase protein. Nucleic Acids Res. 1991 Dec 25;19(24):6691–6698. doi: 10.1093/nar/19.24.6691. [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. Williams K. R., Konigsberg W. H. Identification of amino acid residues at interface of protein-nucleic acid complexes by photochemical cross-linking. Methods Enzymol. 1991;208:516–539. doi: 10.1016/0076-6879(91)08027-f. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Woerner A. M., Marcus-Sekura C. J. Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. Nucleic Acids Res. 1993 Jul 25;21(15):3507–3511. doi: 10.1093/nar/21.15.3507. [DOI] [PMC free article] [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., Groeneger A. A., Plasterk R. H. Mutational analysis of the integrase protein of human immunodeficiency virus type 2. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9598–9602. doi: 10.1073/pnas.89.20.9598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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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