Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Jul 25;19(14):3821–3827. doi: 10.1093/nar/19.14.3821

DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2.

D C van Gent 1, Y Elgersma 1, M W Bolk 1, C Vink 1, R H Plasterk 1
PMCID: PMC328469  PMID: 1861975

Abstract

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

Full text

PDF
3825

Images in this article

3823Image
on p.3823
3824Image
on p.3824
3824Image
on p.3824
3825Image
on p.3825
3825Image
on p.3825
3826Image
on p.3826

Selected References

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

  1. Basu S., Varmus H. E. Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites. J Virol. 1990 Nov;64(11):5617–5625. doi: 10.1128/jvi.64.11.5617-5625.1990. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  2. Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Donehower L. A. Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol. J Virol. 1988 Nov;62(11):3958–3964. doi: 10.1128/jvi.62.11.3958-3964.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Shih C. C., Stoye J. P., Coffin J. M. Highly preferred targets for retrovirus integration. Cell. 1988 May 20;53(4):531–537. doi: 10.1016/0092-8674(88)90569-7. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Vink C., van Gent D. C., Plasterk R. H. Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells. J Virol. 1990 Oct;64(10):5219–5222. doi: 10.1128/jvi.64.10.5219-5222.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]

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

RESOURCES