title: 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.
authors.affiliation: Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111, USA.
authors.forename: Jizu
authors.initials: J
authors.lastname: Yi
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.forename: Mark D
authors.initials: MD
authors.lastname: Andrake
authors.forename: Roland L
authors.initials: RL
authors.lastname: Dunbrack
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
authors.forename: Anna Marie
authors.initials: AM
authors.lastname: Skalka
mesh_terms: D000409:Alanine / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D019427:HIV Integrase / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D007532:Isoleucine; D019427:HIV Integrase; D000409:Alanine