Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Virology logoLink to Journal of Virology
. 1997 Feb;71(2):1025–1035. doi: 10.1128/jvi.71.2.1025-1035.1997

Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions.

M Balakrishnan 1, C B Jonsson 1
PMCID: PMC191153  PMID: 8995622

Abstract

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.

Full Text

The Full Text of this article is available as a PDF (708.6 KB).

Selected References

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

  1. Balakrishnan M., Zastrow D., Jonsson C. B. Catalytic activities of the human T-cell leukemia virus type II integrase. Virology. 1996 May 1;219(1):77–86. doi: 10.1006/viro.1996.0224. [DOI] [PubMed] [Google Scholar]
  2. Brown P. O. Integration of retroviral DNA. Curr Top Microbiol Immunol. 1990;157:19–48. doi: 10.1007/978-3-642-75218-6_2. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Donzella G. A., Jonsson C. B., Roth M. J. Influence of substrate structure on disintegration activity of Moloney murine leukemia virus integrase. J Virol. 1993 Dec;67(12):7077–7087. doi: 10.1128/jvi.67.12.7077-7087.1993. [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. Fitzgerald M. L., Vora A. C., Zeh W. G., Grandgenett D. P. Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase. J Virol. 1992 Nov;66(11):6257–6263. doi: 10.1128/jvi.66.11.6257-6263.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hong T., Murphy E., Groarke J., Drlica K. Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides. J Virol. 1993 Feb;67(2):1127–1131. doi: 10.1128/jvi.67.2.1127-1131.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jonsson C. B., Donzella G. A., Roth M. J. Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli. J Biol Chem. 1993 Jan 15;268(2):1462–1469. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Katz R. A., Skalka A. M. The retroviral enzymes. Annu Rev Biochem. 1994;63:133–173. doi: 10.1146/annurev.bi.63.070194.001025. [DOI] [PubMed] [Google Scholar]
  17. 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]
  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. Kitamura Y., Lee Y. M., Coffin J. M. Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5532–5536. doi: 10.1073/pnas.89.12.5532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  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. 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]
  23. Mizuuchi K. Polynucleotidyl transfer reactions in transpositional DNA recombination. J Biol Chem. 1992 Oct 25;267(30):21273–21276. [PubMed] [Google Scholar]
  24. Pardi D., Switzer W. M., Hadlock K. G., Kaplan J. E., Lal R. B., Folks T. M. Complete nucleotide sequence of an Amerindian human T-cell lymphotropic virus type II (HTLV-II) isolate: identification of a variant HTLV-II subtype b from a Guaymi Indian. J Virol. 1993 Aug;67(8):4659–4664. doi: 10.1128/jvi.67.8.4659-4664.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pemberton I. K., Buckle M., Buc H. The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits a marked preference for Mn(II) rather than Mg(II). J Biol Chem. 1996 Jan 19;271(3):1498–1506. doi: 10.1074/jbc.271.3.1498. [DOI] [PubMed] [Google Scholar]
  26. Pryciak P. M., Sil A., Varmus H. E. Retroviral integration into minichromosomes in vitro. EMBO J. 1992 Jan;11(1):291–303. doi: 10.1002/j.1460-2075.1992.tb05052.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pryciak P. M., Varmus H. E. Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Cell. 1992 May 29;69(5):769–780. doi: 10.1016/0092-8674(92)90289-o. [DOI] [PubMed] [Google Scholar]
  28. Ratner L., Philpott T., Trowbridge D. B. Nucleotide sequence analysis of isolates of human T-lymphotropic virus type 1 of diverse geographical origins. AIDS Res Hum Retroviruses. 1991 Nov;7(11):923–941. doi: 10.1089/aid.1991.7.923. [DOI] [PubMed] [Google Scholar]
  29. Reicin A. S., Kalpana G., Paik S., Marmon S., Goff S. Sequences in the human immunodeficiency virus type 1 U3 region required for in vivo and in vitro integration. J Virol. 1995 Sep;69(9):5904–5907. doi: 10.1128/jvi.69.9.5904-5907.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rohdewohld H., Weiher H., Reik W., Jaenisch R., Breindl M. Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites. J Virol. 1987 Feb;61(2):336–343. doi: 10.1128/jvi.61.2.336-343.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  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. 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]
  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. Störmann K. D., Schlecht M. C., Pfaff E. Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1. J Gen Virol. 1995 Jul;76(Pt 7):1651–1663. doi: 10.1099/0022-1317-76-7-1651. [DOI] [PubMed] [Google Scholar]
  38. Vijaya S., Steffen D. L., Robinson H. L. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin. J Virol. 1986 Nov;60(2):683–692. doi: 10.1128/jvi.60.2.683-692.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Yoshinaga T., Fujiwara T. Different roles of bases within the integration signal sequence of human immunodeficiency virus type 1 in vitro. J Virol. 1995 May;69(5):3233–3236. doi: 10.1128/jvi.69.5.3233-3236.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. van den Ent F. M., Vink C., Plasterk R. H. DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein. J Virol. 1994 Dec;68(12):7825–7832. doi: 10.1128/jvi.68.12.7825-7832.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES