Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Sep 3;93(18):9742–9747. doi: 10.1073/pnas.93.18.9742

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

C M Farnet 1, B Wang 1, J R Lipford 1, F D Bushman 1
PMCID: PMC38499  PMID: 8790401

Abstract

To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.

Full text

PDF
9742

Images in this article

9742Fig. 1
on p.9742
9743Fig. 2
on p.9743
9743Fig. 3
on p.9743

Selected References

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

  1. 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]
  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. 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]
  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. Carteau S., Mouscadet J. F., Goulaouic H., Subra F., Auclair C. Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction. Biochem Biophys Res Commun. 1993 May 14;192(3):1409–1414. doi: 10.1006/bbrc.1993.1573. [DOI] [PubMed] [Google Scholar]
  8. Carteau S., Mouscadet J. F., Goulaouic H., Subra F., Auclair C. Inhibitory effect of the polyanionic drug suramin on the in vitro HIV DNA integration reaction. Arch Biochem Biophys. 1993 Sep;305(2):606–610. doi: 10.1006/abbi.1993.1468. [DOI] [PubMed] [Google Scholar]
  9. Chow S. A., Brown P. O. Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates. J Virol. 1994 Jun;68(6):3896–3907. doi: 10.1128/jvi.68.6.3896-3907.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Cushman M., Golebiewski W. M., Pommier Y., Mazumder A., Reymen D., De Clercq E., Graham L., Rice W. G. Cosalane analogues with enhanced potencies as inhibitors of HIV-1 protease and integrase. J Med Chem. 1995 Feb 3;38(3):443–452. doi: 10.1021/jm00003a007. [DOI] [PubMed] [Google Scholar]
  13. Cushman M., Sherman P. Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions. Biochem Biophys Res Commun. 1992 May 29;185(1):85–90. doi: 10.1016/s0006-291x(05)80958-1. [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. Engelman A., Craigie R. Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity. J Virol. 1995 Sep;69(9):5908–5911. doi: 10.1128/jvi.69.9.5908-5911.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Farnet C. M., Haseltine W. A. Integration of human immunodeficiency virus type 1 DNA in vitro. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4164–4168. doi: 10.1073/pnas.87.11.4164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fesen M. R., Kohn K. W., Leteurtre F., Pommier Y. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2399–2403. doi: 10.1073/pnas.90.6.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fesen M. R., Pommier Y., Leteurtre F., Hiroguchi S., Yung J., Kohn K. W. Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE) and related compounds. Biochem Pharmacol. 1994 Aug 3;48(3):595–608. doi: 10.1016/0006-2952(94)90291-7. [DOI] [PubMed] [Google Scholar]
  20. Gallay P., Swingler S., Aiken C., Trono D. HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell. 1995 Feb 10;80(3):379–388. doi: 10.1016/0092-8674(95)90488-3. [DOI] [PubMed] [Google Scholar]
  21. Gallay P., Swingler S., Song J., Bushman F., Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell. 1995 Nov 17;83(4):569–576. doi: 10.1016/0092-8674(95)90097-7. [DOI] [PubMed] [Google Scholar]
  22. Hickman A. B., Palmer I., Engelman A., Craigie R., Wingfield P. Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase. J Biol Chem. 1994 Nov 18;269(46):29279–29287. [PubMed] [Google Scholar]
  23. Higuchi H., Mori K., Kato A., Ohkuma T., Endo T., Kaji H., Kaji A. Antiretroviral activities of anthraquinones and their inhibitory effects on reverse transcriptase. Antiviral Res. 1991 Mar-Apr;15(3):205–216. doi: 10.1016/0166-3542(91)90067-2. [DOI] [PubMed] [Google Scholar]
  24. Jenkins T. M., Hickman A. B., Dyda F., Ghirlando R., Davies D. R., Craigie R. Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6057–6061. doi: 10.1073/pnas.92.13.6057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. LaFemina R. L., Graham P. L., LeGrow K., Hastings J. C., Wolfe A., Young S. D., Emini E. A., Hazuda D. J. Inhibition of human immunodeficiency virus integrase by bis-catechols. Antimicrob Agents Chemother. 1995 Feb;39(2):320–324. doi: 10.1128/aac.39.2.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lipford J. R., Worland S. T., Farnet C. M. Nucleotide binding by the HIV-1 integrase protein in vitro. J Acquir Immune Defic Syndr. 1994 Dec;7(12):1215–1223. [PubMed] [Google Scholar]
  28. Mazumder A., Cooney D., Agbaria R., Gupta M., Pommier Y. Inhibition of human immunodeficiency virus type 1 integrase by 3'-azido-3'-deoxythymidylate. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5771–5775. doi: 10.1073/pnas.91.13.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mazumder A., Gazit A., Levitzki A., Nicklaus M., Yung J., Kohlhagen G., Pommier Y. Effects of tyrphostins, protein kinase inhibitors, on human immunodeficiency virus type 1 integrase. Biochemistry. 1995 Nov 21;34(46):15111–15122. doi: 10.1021/bi00046a018. [DOI] [PubMed] [Google Scholar]
  30. Mazumder A., Gupta M., Perrin D. M., Sigman D. S., Rabinovitz M., Pommier Y. Inhibition of human immunodeficiency virus type 1 integrase by a hydrophobic cation: the phenanthroline-cuprous complex. AIDS Res Hum Retroviruses. 1995 Jan;11(1):115–125. doi: 10.1089/aid.1995.11.115. [DOI] [PubMed] [Google Scholar]
  31. Mazumder A., Raghavan K., Weinstein J., Kohn K. W., Pommier Y. Inhibition of human immunodeficiency virus type-1 integrase by curcumin. Biochem Pharmacol. 1995 Apr 18;49(8):1165–1170. doi: 10.1016/0006-2952(95)98514-a. [DOI] [PubMed] [Google Scholar]
  32. Miller M. D., Bor Y. C., Bushman F. Target DNA capture by HIV-1 integration complexes. Curr Biol. 1995 Sep 1;5(9):1047–1056. doi: 10.1016/s0960-9822(95)00209-0. [DOI] [PubMed] [Google Scholar]
  33. Mitsuya H., Looney D. J., Kuno S., Ueno R., Wong-Staal F., Broder S. Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells. Science. 1988 Apr 29;240(4852):646–649. doi: 10.1126/science.2452480. [DOI] [PubMed] [Google Scholar]
  34. Puras Lutzke R. A., Eppens N. A., Weber P. A., Houghten R. A., Plasterk R. H. Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11456–11460. doi: 10.1073/pnas.92.25.11456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schinazi R. F., Chu C. K., Babu J. R., Oswald B. J., Saalmann V., Cannon D. L., Eriksson B. F., Nasr M. Anthraquinones as a new class of antiviral agents against human immunodeficiency virus. Antiviral Res. 1990 May;13(5):265–272. doi: 10.1016/0166-3542(90)90071-e. [DOI] [PubMed] [Google Scholar]
  36. Schols D., Baba M., Pauwels R., Desmyter J., De Clercq E. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor. Proc Natl Acad Sci U S A. 1989 May;86(9):3322–3326. doi: 10.1073/pnas.86.9.3322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES