Abstract
We have characterized a murine monoclonal antibody (MAb 35), which was raised against human immunodeficiency virus type 1 (HIV-1) integration protein (IN), and the corresponding Fab 35. Although MAb 35 does not inhibit HIV-1 IN, Fab 35 does. MAb 35 (and Fab 35) binds to an epitope in the C-terminal region of HIV-1 IN. Fab 35 inhibits 3'-end processing, strand transfer, and disintegration; however, DNA binding is not affected. The available data suggest that Fab 35 inhibits enzymatic activities of IN by interfering with the ability of IN to form multimers that are enzymatically active. This implies that the C-terminal region of HIV-1 IN participates in interactions that are essential for the multimerization of IN. Titration of the various IN-mediated enzymatic activities suggests that different degrees of multimerization are required for different activities of HIV-1 IN.
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- Andrake M. D., Skalka A. M. Multimerization determinants reside in both the catalytic core and C terminus of avian sarcoma virus integrase. J Biol Chem. 1995 Dec 8;270(49):29299–29306. doi: 10.1074/jbc.270.49.29299. [DOI] [PubMed] [Google Scholar]
- Biocca S., Neuberger M. S., Cattaneo A. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 1990 Jan;9(1):101–108. doi: 10.1002/j.1460-2075.1990.tb08085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biocca S., Pierandrei-Amaldi P., Cattaneo A. Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of xenopus oocytes. Biochem Biophys Res Commun. 1993 Dec 15;197(2):422–427. doi: 10.1006/bbrc.1993.2496. [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. 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]
- Bujacz G., Jaskólski M., Alexandratos J., Wlodawer A., Merkel G., Katz R. A., Skalka A. M. High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J Mol Biol. 1995 Oct 20;253(2):333–346. doi: 10.1006/jmbi.1995.0556. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Clackson T., Hoogenboom H. R., Griffiths A. D., Winter G. Making antibody fragments using phage display libraries. Nature. 1991 Aug 15;352(6336):624–628. doi: 10.1038/352624a0. [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]
- Drelich M., Wilhelm R., Mous J. Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology. 1992 Jun;188(2):459–468. doi: 10.1016/0042-6822(92)90499-f. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Engelman A., Craigie R. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J Virol. 1992 Nov;66(11):6361–6369. doi: 10.1128/jvi.66.11.6361-6369.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engelman A., Englund G., Orenstein J. M., Martin M. A., Craigie R. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol. 1995 May;69(5):2729–2736. doi: 10.1128/jvi.69.5.2729-2736.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Essen L. O., Skerra A. Single-step purification of a bacterially expressed antibody Fv fragment by immobilized metal affinity chromatography in the presence of betaine. J Chromatogr A. 1993 Dec 24;657(1):55–61. doi: 10.1016/0021-9673(93)83034-p. [DOI] [PubMed] [Google Scholar]
- 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]
- Fujiwara T., Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Hizi A., Hughes S. H. Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli. Virology. 1988 Dec;167(2):634–638. [PubMed] [Google Scholar]
- Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [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]
- Katzman M., Mack J. P., Skalka A. M., Leis J. A covalent complex between retroviral integrase and nicked substrate DNA. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4695–4699. doi: 10.1073/pnas.88.11.4695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Lodi P. J., Ernst J. A., Kuszewski J., Hickman A. B., Engelman A., Craigie R., Clore G. M., Gronenborn A. M. Solution structure of the DNA binding domain of HIV-1 integrase. Biochemistry. 1995 Aug 8;34(31):9826–9833. doi: 10.1021/bi00031a002. [DOI] [PubMed] [Google Scholar]
- 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]
- Marasco W. A., Haseltine W. A., Chen S. Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889–7893. doi: 10.1073/pnas.90.16.7889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orum H., Andersen P. S., Oster A., Johansen L. K., Riise E., Bjørnvad M., Svendsen I., Engberg J. Efficient method for constructing comprehensive murine Fab antibody libraries displayed on phage. Nucleic Acids Res. 1993 Sep 25;21(19):4491–4498. doi: 10.1093/nar/21.19.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plückthun A., Skerra A. Expression of functional antibody Fv and Fab fragments in Escherichia coli. Methods Enzymol. 1989;178:497–515. doi: 10.1016/0076-6879(89)78036-8. [DOI] [PubMed] [Google Scholar]
- Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [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]
- Shin C. G., Taddeo B., Haseltine W. A., Farnet C. M. Genetic analysis of the human immunodeficiency virus type 1 integrase protein. J Virol. 1994 Mar;68(3):1633–1642. doi: 10.1128/jvi.68.3.1633-1642.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Skerra A. A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene. 1994 Apr 8;141(1):79–84. doi: 10.1016/0378-1119(94)90131-7. [DOI] [PubMed] [Google Scholar]
- Smith G. P. Filamentous phage assembly: morphogenetically defective mutants that do not kill the host. Virology. 1988 Nov;167(1):156–165. doi: 10.1016/0042-6822(88)90065-7. [DOI] [PubMed] [Google Scholar]
- Stevenson M., Haggerty S., Lamonica C. A., Meier C. M., Welch S. K., Wasiak A. J. Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol. 1990 May;64(5):2421–2425. doi: 10.1128/jvi.64.5.2421-2425.1990. [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]
- Vinga-Martins C., Schneider T., Werno A., Roenspeck W., Pauli G., Mueller-Lantzsch N. Mapping of immunodominant epitopes of the HIV-1 and HIV-2 integrase proteins by recombinant proteins and synthetic peptides. AIDS Res Hum Retroviruses. 1992 Jul;8(7):1301–1310. doi: 10.1089/aid.1992.8.1301. [DOI] [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]
- 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]
- Werge T. M., Biocca S., Cattaneo A. Intracellular immunization. Cloning and intracellular expression of a monoclonal antibody to the p21ras protein. FEBS Lett. 1990 Nov 12;274(1-2):193–198. doi: 10.1016/0014-5793(90)81362-r. [DOI] [PubMed] [Google Scholar]
- Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
- Winter G., Griffiths A. D., Hawkins R. E., Hoogenboom H. R. Making antibodies by phage display technology. Annu Rev Immunol. 1994;12:433–455. doi: 10.1146/annurev.iy.12.040194.002245. [DOI] [PubMed] [Google Scholar]
- 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]
- Woerner A. M., Marcus-Sekura C. J. Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. Nucleic Acids Res. 1993 Jul 25;21(15):3507–3511. doi: 10.1093/nar/21.15.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]