Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

The Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Retrovirology (Impact Factor: 4.19). 02/2006; 3(1):34. DOI: 10.1186/1742-4690-3-34
Source: PubMed


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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    • "). Also, a large array of recombinant mutants of HIV integrase have been assayed by SPR for their interaction with DNA (Ramcharan et al., 2006) and the same has been performed for the interaction of HIV Nef mutants with Hck-SH3 (Manninen et al., 1998). Regarding adenoviruses , worth to note is the paper by Alba et al. (2009) in which the adenovirus 5 hexon was point mutated, expressed in intact virions and then assayed for its binding to coagulation factor X. Also, SPR has been instrumental in evaluating the interaction of fiber knob protein mutants with CAR and in the reverse approach, namely the screening of peptides representing the immunoglobulin domain of CAR for their capacity to bind to the fiber knob protein (Kirby et al., 2000). "
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