Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Nov;70(11):7706–7712. doi: 10.1128/jvi.70.11.7706-7712.1996

Human recombinant antibody fragments neutralizing human immunodeficiency virus type 1 reverse transcriptase provide an experimental basis for the structural classification of the DNA polymerase family.

N Gargano 1, S Biocca 1, A Bradbury 1, A Cattaneo 1
PMCID: PMC190840  PMID: 8892891

Abstract

We describe in this paper the binding and biochemical properties of two human antibody fragments directed against the human immunodeficiency virus type 1 reverse transcriptase (RT). These fragments were isolated from a synthetic combinatorial library of human Fab antibody fragments displayed on the surface of filamentous phage. The antibody fragments were selected by using recombinant heterodimeric human immunodeficiency virus type 1 RT purified from insect cells as a solid-phase selector. This procedure led to the isolation of two antibody fragments that completely neutralize the RNA-dependent DNA polymerase activity of RT at nanomolar concentrations. Both antibody fragments bind only to the enzymatically active form of the RT. The inhibitory activity of the anti-RT antibody fragments is competitive with respect to the template primer. The antibody fragments also neutralize the activities of RTs from avian and murine retroviruses and of DNA polymerases of prokaryotic origin as well as human DNA polymerase alpha. Thus, the antibody fragments selected and characterized in this study appear to recognize a structural fold that is common to the different DNA polymerases and necessary for their activity. The results provide an immunological experimental basis for a purely structural and evolutionary classification of the polymerase family.

Full Text

The Full Text of this article is available as a PDF (279.8 KB).

Selected References

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

  1. Beard W. A., Wilson S. H. Site-directed mutagenesis of HIV reverse transcriptase to probe enzyme processivity and drug binding. Curr Opin Biotechnol. 1994 Aug;5(4):414–421. doi: 10.1016/0958-1669(94)90051-5. [DOI] [PubMed] [Google Scholar]
  2. Biocca S., Cattaneo A. Intracellular immunization: antibody targeting to subcellular compartments. Trends Cell Biol. 1995 Jun;5(6):248–252. doi: 10.1016/s0962-8924(00)89019-4. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Davies J. F., 2nd, Almassy R. J., Hostomska Z., Ferre R. A., Hostomsky Z. 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell. 1994 Mar 25;76(6):1123–1133. doi: 10.1016/0092-8674(94)90388-3. [DOI] [PubMed] [Google Scholar]
  5. De Clercq E. HIV resistance to reverse transcriptase inhibitors. Biochem Pharmacol. 1994 Jan 20;47(2):155–169. doi: 10.1016/0006-2952(94)90001-9. [DOI] [PubMed] [Google Scholar]
  6. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  7. Duan L., Bagasra O., Laughlin M. A., Oakes J. W., Pomerantz R. J. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5075–5079. doi: 10.1073/pnas.91.11.5075. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  8. Esnouf R., Ren J., Ross C., Jones Y., Stammers D., Stuart D. Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. Nat Struct Biol. 1995 Apr;2(4):303–308. doi: 10.1038/nsb0495-303. [DOI] [PubMed] [Google Scholar]
  9. Ferns R. B., Partridge J. C., Tisdale M., Hunt N., Tedder R. S. Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products. AIDS Res Hum Retroviruses. 1991 Mar;7(3):307–313. doi: 10.1089/aid.1991.7.307. [DOI] [PubMed] [Google Scholar]
  10. Ferris A. L., Hizi A., Showalter S. D., Pichuantes S., Babe L., Craik C. S., Hughes S. H. Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure. Virology. 1990 Apr;175(2):456–464. doi: 10.1016/0042-6822(90)90430-y. [DOI] [PubMed] [Google Scholar]
  11. Gait M. J., Karn J. Progress in anti-HIV structure-based drug design. Trends Biotechnol. 1995 Oct;13(10):430–438. doi: 10.1016/S0167-7799(00)88998-2. [DOI] [PubMed] [Google Scholar]
  12. Georgiadis M. M., Jessen S. M., Ogata C. M., Telesnitsky A., Goff S. P., Hendrickson W. A. Mechanistic implications from the structure of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase. Structure. 1995 Sep 15;3(9):879–892. doi: 10.1016/S0969-2126(01)00223-4. [DOI] [PubMed] [Google Scholar]
  13. Griffiths A. D., Williams S. C., Hartley O., Tomlinson I. M., Waterhouse P., Crosby W. L., Kontermann R. E., Jones P. T., Low N. M., Allison T. J. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 1994 Jul 15;13(14):3245–3260. doi: 10.1002/j.1460-2075.1994.tb06626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hizi A., Tal R., Shaharabany M., Loya S. Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. J Biol Chem. 1991 Apr 5;266(10):6230–6239. [PubMed] [Google Scholar]
  15. Hughes S. H., Hostomsky Z., Le Grice S. F., Lentz K., Arnold E. What is the orientation of DNA polymerases on their templates? J Virol. 1996 May;70(5):2679–2683. doi: 10.1128/jvi.70.5.2679-2683.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim Y., Eom S. H., Wang J., Lee D. S., Suh S. W., Steitz T. A. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612–616. doi: 10.1038/376612a0. [DOI] [PubMed] [Google Scholar]
  17. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  18. Maciejewski J. P., Weichold F. F., Young N. S., Cara A., Zella D., Reitz M. S., Jr, Gallo R. C. Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro. Nat Med. 1995 Jul;1(7):667–673. doi: 10.1038/nm0795-667. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Marks J. D., Hoogenboom H. R., Bonnert T. P., McCafferty J., Griffiths A. D., Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. doi: 10.1016/0022-2836(91)90498-u. [DOI] [PubMed] [Google Scholar]
  21. Mhashilkar A. M., Bagley J., Chen S. Y., Szilvay A. M., Helland D. G., Marasco W. A. Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. EMBO J. 1995 Apr 3;14(7):1542–1551. doi: 10.1002/j.1460-2075.1995.tb07140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Patel P. H., Jacobo-Molina A., Ding J., Tantillo C., Clark A. D., Jr, Raag R., Nanni R. G., Hughes S. H., Arnold E. Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. Biochemistry. 1995 Apr 25;34(16):5351–5363. doi: 10.1021/bi00016a006. [DOI] [PubMed] [Google Scholar]
  23. Ren J., Esnouf R., Garman E., Somers D., Ross C., Kirby I., Keeling J., Darby G., Jones Y., Stuart D. High resolution structures of HIV-1 RT from four RT-inhibitor complexes. Nat Struct Biol. 1995 Apr;2(4):293–302. doi: 10.1038/nsb0495-293. [DOI] [PubMed] [Google Scholar]
  24. Ren J., Esnouf R., Hopkins A., Ross C., Jones Y., Stammers D., Stuart D. The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design. Structure. 1995 Sep 15;3(9):915–926. doi: 10.1016/S0969-2126(01)00226-X. [DOI] [PubMed] [Google Scholar]
  25. Restle T., Pawlita M., Sczakiel G., Müller B., Goody R. S. Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies. J Biol Chem. 1992 Jul 25;267(21):14654–14661. [PubMed] [Google Scholar]
  26. Richardson J. H., Marasco W. A. Intracellular antibodies: development and therapeutic potential. Trends Biotechnol. 1995 Aug;13(8):306–310. doi: 10.1016/S0167-7799(00)88970-2. [DOI] [PubMed] [Google Scholar]
  27. Richman D. D. HIV drug resistance. Annu Rev Pharmacol Toxicol. 1993;33:149–164. doi: 10.1146/annurev.pa.33.040193.001053. [DOI] [PubMed] [Google Scholar]
  28. Sawaya M. R., Pelletier H., Kumar A., Wilson S. H., Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science. 1994 Jun 24;264(5167):1930–1935. doi: 10.1126/science.7516581. [DOI] [PubMed] [Google Scholar]
  29. Tantillo C., Ding J., Jacobo-Molina A., Nanni R. G., Boyer P. L., Hughes S. H., Pauwels R., Andries K., Janssen P. A., Arnold E. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J Mol Biol. 1994 Oct 28;243(3):369–387. doi: 10.1006/jmbi.1994.1665. [DOI] [PubMed] [Google Scholar]
  30. Tisdale M., Ertl P., Larder B. A., Purifoy D. J., Darby G., Powell K. L. Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function. J Virol. 1988 Oct;62(10):3662–3667. doi: 10.1128/jvi.62.10.3662-3667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tomlinson I. M., Walter G., Marks J. D., Llewelyn M. B., Winter G. The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. J Mol Biol. 1992 Oct 5;227(3):776–798. doi: 10.1016/0022-2836(92)90223-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Wu J., Amandoron E., Li X., Wainberg M. A., Parniak M. A. Monoclonal antibody-mediated inhibition of HIV-1 reverse transcriptase polymerase activity. Interaction with a possible deoxynucleoside triphosphate binding domain. J Biol Chem. 1993 May 15;268(14):9980–9985. [PubMed] [Google Scholar]
  34. Yu M., Poeschla E., Wong-Staal F. Progress towards gene therapy for HIV infection. Gene Ther. 1994 Jan;1(1):13–26. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES