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  Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies

Restle, T., Pawlita, M., Sczakiel, G., Müller, B., & Goody, R. S. (1992). Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies. The Journal of Biological Chemistry, 267(21), 14654-14661. Retrieved from http://www.jbc.org/cgi/content/abstract/267/21/14654.

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Restle, Tobias1, 2, Author              
Pawlita, Michael, Author
Sczakiel, Georg1, Author              
Müller, Barbara1, Author              
Goody, Roger S.1, Author              
Affiliations:
1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              
2Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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 Abstract: The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.

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Language(s): eng - English
 Dates: 1991-09-031992-07-25
 Publication Status: Published in print
 Pages: 8
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 Table of Contents: -
 Rev. Type: Peer
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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: - Volume / Issue: 267 (21) Sequence Number: - Start / End Page: 14654 - 14661 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1