Mapping the Epitope of an Inhibitory Monoclonal Antibody to the C-terminal DNA-binding Domain of HIV-1 Integrase

Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2002; 277(14):12164-74. DOI: 10.1074/jbc.M105072200
Source: PubMed


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.

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    • "The binding of mAb33 was previously reported to reduce mobility of contiguous residues on the surface of the β-strands of the isolated CTD [28]. It was proposed that these residues, F223, R224, and Y226 on β1, K244 on β2, and I267 and I268 on β5 (Figure 1A) comprise the mAb33 epitope. "
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    ABSTRACT: To further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing.
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