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

Abstract

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 DNAduplex (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.