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J Virol. 1997 Jul; 71(7): 5382–5390.
PMCID: PMC191777
PMID: 9188609

Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition.

M D Miller, C M Farnet, and F D Bushman
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Abstract

We have investigated the organization and function of human immunodeficiency virus type 1 (HIV-1) preintegration complexes (PICs), the large nucleoprotein particles that carry out cDNA integration in vivo. PICs can be isolated from HIV-1-infected cells, and such particles are capable of carrying out integration reactions in vitro. We find that although the PICs are large, the cDNA must be condensed to fit into the measured volume. The ends of the cDNA are probably linked by a protein bridge, since coordinated joining of the two ends is not disrupted by cleaving the cDNA internally with a restriction enzyme. cDNA ends in PICs were protected from digestion by added exonucleases, probably due to binding of proteins. The intervening cDNA, in contrast, was susceptible to attack by endonucleases. Previous work has established that the virus-encoded integrase protein is present in PICs, and we have reported recently that the host protein HMG I(Y) is also present. Here we report that the viral matrix and reverse transcriptase (RT) proteins also cofractionated with PICs through several steps whereas capsid and nucleocapsid proteins dissociated. These data support a model of PIC organization in which the cDNA is condensed in a partially disassembled remnant of the viral core, with proteins tightly associated at the apposed cDNA ends but loosely associated with the intervening cDNA. In characterizing the structure of the cDNA ends, we found that the U5 DNA ends created by RT were ragged, probably due to the terminal transferase activity of RT. Only molecules correctly cleaved by integrase protein at the 3' ends were competent to integrate, suggesting that one role for terminal cleavage by integrase may be to create a defined end at otherwise heterogeneous cDNA termini.

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Selected References

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  • Aldaz H, Schuster E, Baker TA. The interwoven architecture of the Mu transposase couples DNA synapsis to catalysis. Cell. 1996 Apr 19;85(2):257–269. [PubMed] [Google Scholar]
  • Baker TA, Kremenstova E, Luo L. Complete transposition requires four active monomers in the mu transposase tetramer. Genes Dev. 1994 Oct 15;8(20):2416–2428. [PubMed] [Google Scholar]
  • Baker TA, Mizuuchi M, Savilahti H, Mizuuchi K. Division of labor among monomers within the Mu transposase tetramer. Cell. 1993 Aug 27;74(4):723–733. [PubMed] [Google Scholar]
  • Bowerman B, Brown PO, Bishop JM, Varmus HE. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. [PubMed] [Google Scholar]
  • Brown PO, Bowerman B, Varmus HE, Bishop JM. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. [PubMed] [Google Scholar]
  • Brown PO, Bowerman B, Varmus HE, Bishop JM. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. [PMC free article] [PubMed] [Google Scholar]
  • Bukrinsky MI, Sharova N, McDonald TL, Pushkarskaya T, Tarpley WG, 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. [PMC free article] [PubMed] [Google Scholar]
  • Bushman FD, Miller MD. Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J Virol. 1997 Jan;71(1):458–464. [PMC free article] [PubMed] [Google Scholar]
  • Bushman FD, 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. [PMC free article] [PubMed] [Google Scholar]
  • Bushman FD, Fujiwara T, Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. [PubMed] [Google Scholar]
  • Charneau P, Clavel F. A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J Virol. 1991 May;65(5):2415–2421. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Eichinger DJ, Boeke JD. A specific terminal structure is required for Ty1 transposition. Genes Dev. 1990 Mar;4(3):324–330. [PubMed] [Google Scholar]
  • Ellison V, Brown PO. 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. [PMC free article] [PubMed] [Google Scholar]
  • Ellison V, Gerton J, Vincent KA, Brown PO. 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. [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • Engelman A, Bushman FD, 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. [PMC free article] [PubMed] [Google Scholar]
  • Farnet CM, Bushman FD. HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro. Cell. 1997 Feb 21;88(4):483–492. [PubMed] [Google Scholar]
  • Farnet CM, Wang B, Lipford JR, Bushman FD. Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9742–9747. [PMC free article] [PubMed] [Google Scholar]
  • Farnet CM, Bushman FD. HIV cDNA integration: molecular biology and inhibitor development. AIDS. 1996;10 (Suppl A):S3–11. [PubMed] [Google Scholar]
  • Farnet CM, Haseltine WA. Integration of human immunodeficiency virus type 1 DNA in vitro. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4164–4168. [PMC free article] [PubMed] [Google Scholar]
  • Farnet CM, Haseltine WA. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol. 1991 Apr;65(4):1910–1915. [PMC free article] [PubMed] [Google Scholar]
  • Fujiwara T, Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Gelderblom HR, Hausmann EH, Ozel M, Pauli G, Koch MA. Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology. 1987 Jan;156(1):171–176. [PubMed] [Google Scholar]
  • Grandgenett DP, Inman RB, Vora AC, Fitzgerald ML. Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase. J Virol. 1993 May;67(5):2628–2636. [PMC free article] [PubMed] [Google Scholar]
  • Hansen MS, Bushman FD. Human immunodeficiency virus type 2 preintegration complexes: activities in vitro and response to inhibitors. J Virol. 1997 Apr;71(4):3351–3356. [PMC free article] [PubMed] [Google Scholar]
  • Jones KS, Coleman J, Merkel GW, Laue TM, Skalka AM. Retroviral integrase functions as a multimer and can turn over catalytically. J Biol Chem. 1992 Aug 15;267(23):16037–16040. [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Katz RA, Merkel G, Kulkosky J, Leis J, Skalka AM. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. [PubMed] [Google Scholar]
  • Katzman M, Katz RA, Skalka AM, 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. [PMC free article] [PubMed] [Google Scholar]
  • Lavoie BD, Chan BS, Allison RG, Chaconas G. Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J. 1991 Oct;10(10):3051–3059. [PMC free article] [PubMed] [Google Scholar]
  • Miller MD, Wang B, Bushman FD. Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro. J Virol. 1995 Jun;69(6):3938–3944. [PMC free article] [PubMed] [Google Scholar]
  • Mizuuchi M, Baker TA, Mizuuchi K. Assembly of the active form of the transposase-Mu DNA complex: a critical control point in Mu transposition. Cell. 1992 Jul 24;70(2):303–311. [PubMed] [Google Scholar]
  • Patel PH, Preston BD. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):549–553. [PMC free article] [PubMed] [Google Scholar]
  • Pauza CD. Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination. Virology. 1990 Dec;179(2):886–889. [PubMed] [Google Scholar]
  • Peliska JA, Benkovic SJ. Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Science. 1992 Nov 13;258(5085):1112–1118. [PubMed] [Google Scholar]
  • Roth MJ, Schwartzberg PL, Goff SP. 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. [PubMed] [Google Scholar]
  • Savilahti H, Mizuuchi K. Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. Cell. 1996 Apr 19;85(2):271–280. [PubMed] [Google Scholar]
  • Sherman PA, Fyfe JA. 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. [PMC free article] [PubMed] [Google Scholar]
  • Siegel LM, Monty KJ. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. [PubMed] [Google Scholar]
  • van Gent DC, Vink C, Groeneger AA, Plasterk RH. Complementation between HIV integrase proteins mutated in different domains. EMBO J. 1993 Aug;12(8):3261–3267. [PMC free article] [PubMed] [Google Scholar]
  • Vink C, Plasterk RH. The human immunodeficiency virus integrase protein. Trends Genet. 1993 Dec;9(12):433–438. [PubMed] [Google Scholar]
  • Vink C, Lutzke RA, Plasterk RH. 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. [PMC free article] [PubMed] [Google Scholar]
  • Vora AC, McCord M, Fitzgerald ML, Inman RB, Grandgenett DP. Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase. Nucleic Acids Res. 1994 Oct 25;22(21):4454–4461. [PMC free article] [PubMed] [Google Scholar]
  • Yang JY, Jayaram M, Harshey RM. Positional information within the Mu transposase tetramer: catalytic contributions of individual monomers. Cell. 1996 May 3;85(3):447–455. [PubMed] [Google Scholar]
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