Mapping the epitope of an inhibitory monoclonal antibody to the C-terminal DNA-binding domain of HIV-1 integrase.
ABSTRACT Integrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, monoclonal antibody (mAb) 33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here, we show that mAb33 is a strong inhibitor of IN catalytic activity, whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 had no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 did inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevented DNA binding to the isolated C-terminal domain of IN. Results from size-exclusion chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses revealed that multiple Fab33 small middle dotIN C-terminal domain complexes exist in solution. Studies using heteronuclear NMR showed a steep decrease in (1)H-(15)N cross-peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala(239) and Ile(251) are buried in the interior of the domain, whereas the remaining residues (Phe(223), Arg(224), Tyr(226), Lys(244), Ile(267), and Ile(268)) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggested a structure for the antibody-antigen complex that is consistent with our experimental data and suggested a potential target for anti-AIDS drug design.
Full-textDOI: · Available from: Anna marie Skalka, May 24, 2015
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ABSTRACT: Living organisms evolved by developing the capacity to convert an available form of energy into a useful form of energy; how well they thrive depends on how effectively a needed conversion is performed. http://link.springer.com/book/10.1007/978-3-0348-9179-0/page/1Bioelectrochemistry: Principles and Practice: Vol. 5, Bioelectrochemistry of Biomacromolecules, Edited by Giorgio Lenaz, Giulio Milazzo, 01/1997: chapter 2: pages 105-182; Birkhäuser Verlag AG., ISBN: 978-3-0348-9936-9 (Print) 978-3-0348-9179-0 (Online)
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ABSTRACT: AIDS is currently treated with a combination therapy of reverse transcriptase and protease inhibitors. Recently, the FDA approved a drug targeting HIV-1 entry into cells. There are currently no FDA approved drugs targeting HIV-1 integrase, though many scientists and drug companies are actively in pursuit of clinically useful integrase inhibitors. The objective of this review is to provide an update on integrase inhibitors reported in the last two years, including two novel inhibitors in early clinical trials, recently developed hydroxylated aromatics, natural products, peptide, antibody and oligonucleotide inhibitors. Additionally, the proposed mechanism of diketo acid inhibition is reviewed.Current Topics in Medicinal Chemistry 06/2004; 4(10):1059-1077. DOI:10.2174/1568026043388394 · 3.45 Impact Factor