Abstract
HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5′-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3′-processing inhibitors, indicating that cross-linking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3′-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas 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 cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA.
Footnotes
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This research was supported by the National Institutes of Health Intramural Program, Center for Cancer Research, National Cancer Institute.
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A.A.J. and C.M. contributed equally to this work
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.030817.
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ABBREVIATIONS: LTR, long terminal repeat; 3′-P, 3′-processing; MOPS, 4-morpholinepropanesulfonic acid; DKA, diketo acid; l-CA, l-chicoric acid repeat; ST, strand transfer.
- Received September 13, 2006.
- Accepted December 14, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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