Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Oct 11;22(20):4125–4131. doi: 10.1093/nar/22.20.4125

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

R A Lutzke 1, C Vink 1, R H Plasterk 1
PMCID: PMC331899  PMID: 7937137

Abstract

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.

Full text

PDF
4125

Images in this article

4127Image
on p.4127
4128Image
on p.4128
4128Image
on p.4128
4130Image
on p.4130

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrésson O. S., Elser J. E., Tobin G. J., Greenwood J. D., Gonda M. A., Georgsson G., Andrésdóttir V., Benediktsdóttir E., Carlsdóttir H. M., Mäntylä E. O. Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus. Virology. 1993 Mar;193(1):89–105. doi: 10.1006/viro.1993.1106. [DOI] [PubMed] [Google Scholar]
  3. Beese L. S., Steitz T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. doi: 10.1002/j.1460-2075.1991.tb07917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bukrinsky M. I., Sharova N., McDonald T. L., Pushkarskaya T., Tarpley W. G., Stevenson M. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6125–6129. doi: 10.1073/pnas.90.13.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burke C. J., Sanyal G., Bruner M. W., Ryan J. A., LaFemina R. L., Robbins H. L., Zeft A. S., Middaugh C. R., Cordingley M. G. Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase. J Biol Chem. 1992 May 15;267(14):9639–9644. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Farnet C. M., Haseltine W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol. 1991 Apr;65(4):1910–1915. doi: 10.1128/jvi.65.4.1910-1915.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Fomsgaard A., Hirsch V. M., Allan J. S., Johnson P. R. A highly divergent proviral DNA clone of SIV from a distinct species of African green monkey. Virology. 1991 May;182(1):397–402. doi: 10.1016/0042-6822(91)90689-9. [DOI] [PubMed] [Google Scholar]
  15. Garvey K. J., Oberste M. S., Elser J. E., Braun M. J., Gonda M. A. Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus. Virology. 1990 Apr;175(2):391–409. doi: 10.1016/0042-6822(90)90424-p. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Guyader M., Emerman M., Sonigo P., Clavel F., Montagnier L., Alizon M. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature. 1987 Apr 16;326(6114):662–669. doi: 10.1038/326662a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Lapadat-Tapolsky M., De Rocquigny H., Van Gent D., Roques B., Plasterk R., Darlix J. L. Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. Nucleic Acids Res. 1993 Feb 25;21(4):831–839. doi: 10.1093/nar/21.4.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Malik K. T., Even J., Karpas A. Molecular cloning and complete nucleotide sequence of an adult T cell leukaemia virus/human T cell leukaemia virus type I (ATLV/HTLV-I) isolate of Caribbean origin: relationship to other members of the ATLV/HTLV-I subgroup. J Gen Virol. 1988 Jul;69(Pt 7):1695–1710. doi: 10.1099/0022-1317-69-7-1695. [DOI] [PubMed] [Google Scholar]
  27. Moore R., Dixon M., Smith R., Peters G., Dickson C. Complete nucleotide sequence of a milk-transmitted mouse mammary tumor virus: two frameshift suppression events are required for translation of gag and pol. J Virol. 1987 Feb;61(2):480–490. doi: 10.1128/jvi.61.2.480-490.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  32. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Shimotohno K., Takahashi Y., Shimizu N., Gojobori T., Golde D. W., Chen I. S., Miwa M., Sugimura T. Complete nucleotide sequence of an infectious clone of human T-cell leukemia virus type II: an open reading frame for the protease gene. Proc Natl Acad Sci U S A. 1985 May;82(10):3101–3105. doi: 10.1073/pnas.82.10.3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  37. Skalka A. M. Retroviral DNA integration: lessons for transposon shuffling. Gene. 1993 Dec 15;135(1-2):175–182. doi: 10.1016/0378-1119(93)90063-9. [DOI] [PubMed] [Google Scholar]
  38. Sonigo P., Barker C., Hunter E., Wain-Hobson S. Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus. Cell. 1986 May 9;45(3):375–385. doi: 10.1016/0092-8674(86)90323-5. [DOI] [PubMed] [Google Scholar]
  39. Stephens R. M., Casey J. W., Rice N. R. Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. Science. 1986 Feb 7;231(4738):589–594. doi: 10.1126/science.3003905. [DOI] [PubMed] [Google Scholar]
  40. Stevenson M., Haggerty S., Lamonica C. A., Meier C. M., Welch S. K., Wasiak A. J. Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol. 1990 May;64(5):2421–2425. doi: 10.1128/jvi.64.5.2421-2425.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Talbott R. L., Sparger E. E., Lovelace K. M., Fitch W. M., Pedersen N. C., Luciw P. A., Elder J. H. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5743–5747. doi: 10.1073/pnas.86.15.5743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vincent K. A., Ellison V., Chow S. A., Brown P. O. Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations. J Virol. 1993 Jan;67(1):425–437. doi: 10.1128/jvi.67.1.425-437.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Vink C., van der Linden K. H., Plasterk R. H. Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli. J Virol. 1994 Mar;68(3):1468–1474. doi: 10.1128/jvi.68.3.1468-1474.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Whetter L., Archambault D., Perry S., Gazit A., Coggins L., Yaniv A., Clabough D., Dahlberg J., Fuller F., Tronick S. Equine infectious anemia virus derived from a molecular clone persistently infects horses. J Virol. 1990 Dec;64(12):5750–5756. doi: 10.1128/jvi.64.12.5750-5756.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES