Received for publication September 14, 2006.
Revised December 14, 2006.
Accepted for publication December 14, 2006.

PROBING HIV-1 INTEGRASE INHIBITOR BINDING SITES WITH POSITION-SPECIFIC INTEGRASE-DNA CROSSLINKING ASSAYS

Abstract

HIV-1 integrase binds to the ends of the viral cDNA site-specifically. We used two HIV-1 integrase-DNA crosslinking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivity. The disulfide assay probes crosslinking between the integrase residue 148 and the 5’-terminal cytosine (5'-C) of the viral cDNA, and the Schiff base assay probes crosslinking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Crosslinking interference by eight integrase inhibitors shows that the most potent crosslinking inhibitors are 3'-processing inhibitors, indicating that crosslinking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak crosslinking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent crosslinking formation. Four inhibitors that prevented Schiff base crosslinking to the conserved 3'-terminal adenine (3'-A) position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand crosslinking, while the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The crosslinking assays also provide clues to the molecular interactions between integrase and the viral cDNA.

Key words: Structure-activity relationships and modeling, Antiviral drugs