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J. Biol. Chem., Vol. 281, Issue 1, 461-467, January 6, 2006

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Integration Requires a Specific Interaction of the Donor DNA Terminal 5'-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop^*

Allison A. Johnson, Webster Santos, Godwin C. G. Pais¶, Christophe Marchand, Ronak Amin, Terrence R. Burke, Jr.¶, Gregory Verdine, and Yves Pommier1

From the Laboratory of Molecular Pharmacology and ^¶Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.

Received for publication, October 19, 2005

^* This research was supported in part by the Intramural Research Program of NCI, Center for Cancer Research, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 5068, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

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