Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Mar;70(3):1424–1432. doi: 10.1128/jvi.70.3.1424-1432.1996

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

A L Wolfe 1, P J Felock 1, J C Hastings 1, C U Blau 1, D J Hazuda 1
PMCID: PMC189962  PMID: 8627659

Abstract

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.

Full Text

The Full Text of this article is available as a PDF (252.8 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Bukrinsky M., Sharova N., Stevenson M. Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex. J Virol. 1993 Nov;67(11):6863–6865. doi: 10.1128/jvi.67.11.6863-6865.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Cobrinik D., Katz R., Terry R., Skalka A. M., Leis J. Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth. J Virol. 1987 Jun;61(6):1999–2008. doi: 10.1128/jvi.61.6.1999-2008.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Donehower L. A., Varmus H. E. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6461–6465. doi: 10.1073/pnas.81.20.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Dyda F., Hickman A. B., Jenkins T. M., Engelman A., Craigie R., Davies D. R. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science. 1994 Dec 23;266(5193):1981–1986. doi: 10.1126/science.7801124. [DOI] [PubMed] [Google Scholar]
  14. Ellison V., Abrams H., Roe T., Lifson J., Brown P. Human immunodeficiency virus integration in a cell-free system. J Virol. 1990 Jun;64(6):2711–2715. doi: 10.1128/jvi.64.6.2711-2715.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ellison V., Brown P. O. A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7316–7320. doi: 10.1073/pnas.91.15.7316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ellison V., Gerton J., Vincent K. A., Brown P. O. An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer. J Biol Chem. 1995 Feb 17;270(7):3320–3326. doi: 10.1074/jbc.270.7.3320. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Engelman A., Hickman A. B., Craigie R. The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding. J Virol. 1994 Sep;68(9):5911–5917. doi: 10.1128/jvi.68.9.5911-5917.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Englund G., Theodore T. S., Freed E. O., Engelman A., Martin M. A. Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1. J Virol. 1995 May;69(5):3216–3219. doi: 10.1128/jvi.69.5.3216-3219.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Grandgenett D. P., Goodarzi G. Folding of the multidomain human immunodeficiency virus type-I integrase. Protein Sci. 1994 Jun;3(6):888–897. doi: 10.1002/pro.5560030604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Hazuda D. J., Hastings J. C., Wolfe A. L., Emini E. A. A novel assay for the DNA strand-transfer reaction of HIV-1 integrase. Nucleic Acids Res. 1994 Mar 25;22(6):1121–1122. doi: 10.1093/nar/22.6.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Jones K. S., Coleman J., Merkel G. W., Laue T. M., Skalka A. M. Retroviral integrase functions as a multimer and can turn over catalytically. J Biol Chem. 1992 Aug 15;267(23):16037–16040. [PubMed] [Google Scholar]
  27. 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]
  28. Karageorgos L., Li P., Burrell C. Characterization of HIV replication complexes early after cell-to-cell infection. AIDS Res Hum Retroviruses. 1993 Sep;9(9):817–823. doi: 10.1089/aid.1993.9.817. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Lutzke R. A., Vink C., Plasterk R. H. Characterization of the minimal DNA-binding domain of the HIV integrase protein. Nucleic Acids Res. 1994 Oct 11;22(20):4125–4131. doi: 10.1093/nar/22.20.4125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Panganiban A. T., Temin H. M. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Panganiban A. T., Temin H. M. The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature. 1983 Nov 10;306(5939):155–160. doi: 10.1038/306155a0. [DOI] [PubMed] [Google Scholar]
  33. Quinn T. P., Grandgenett D. P. Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration. J Virol. 1988 Jul;62(7):2307–2312. doi: 10.1128/jvi.62.7.2307-2312.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Stevenson M., Stanwick T. L., Dempsey M. P., Lamonica C. A. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J. 1990 May;9(5):1551–1560. doi: 10.1002/j.1460-2075.1990.tb08274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Vink C., Lutzke R. A., Plasterk R. H. Formation of a stable complex between the human immunodeficiency virus integrase protein and viral DNA. Nucleic Acids Res. 1994 Oct 11;22(20):4103–4110. doi: 10.1093/nar/22.20.4103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wiskerchen M., Muesing M. A. Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J Virol. 1995 Jan;69(1):376–386. doi: 10.1128/jvi.69.1.376-386.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]
  41. 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 Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES