Abstract
Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.
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Selected References
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- Baker T. A., Mizuuchi K. DNA-promoted assembly of the active tetramer of the Mu transposase. Genes Dev. 1992 Nov;6(11):2221–2232. doi: 10.1101/gad.6.11.2221. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. 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. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Bushman F. D., Craigie R. Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3458–3462. doi: 10.1073/pnas.89.8.3458. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Echols H. Nucleoprotein structures initiating DNA replication, transcription, and site-specific recombination. J Biol Chem. 1990 Sep 5;265(25):14697–14700. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Mizuuchi M., Baker T. A., 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. doi: 10.1016/0092-8674(92)90104-k. [DOI] [PubMed] [Google Scholar]
- Roe T., Reynolds T. C., Yu G., Brown P. O. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 1993 May;12(5):2099–2108. doi: 10.1002/j.1460-2075.1993.tb05858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]