Effect of DNA Modifications on DNA Processing by HIV-1 Integrase and Inhibitor Binding: ROLE OF DNA BACKBONE FLEXIBILITY AND AN OPEN CATALYTIC SITE

Allison A Johnson,Jane M Sayer,Haruhiko Yagi,Sachindra S Patil,Françoise Debart,Martin A Maier,David R Corey,Jean-Jacques Vasseur,Terrence R Burke,Victor E Marquez,Donald M Jerina,Yves Pommier

doi:10.1074/jbc.m605101200

Abstract

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

Highlights

From the ‡Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, §Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, ¶Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Department of Health and Human Services, Frederick, Maryland 20892, ʈLCOBS UMR 5625 CNRS-Universite Montpellier II, 34095 Montpellier, France, **Isis Pharmaceuticals, Inc., Carlsbad, California 92008, and ‡‡Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235
The hydrocarbon portion of the guanine N-2 adducts lies in the minor groove of the DNA and extends toward the 5Ј or 3Ј terminus of the adducted strand for the trans(S) and trans-(R) adducts, respectively, where S and R refer to the absolute configuration at the point of attachment of the 2-amino group of the hydrocarbon [7, 16] We reported previously that a BaP DE dG adduct in the minor groove of the 3Ј-P site blocked 3Ј-P [7]
These results show that conformational restrictions at the 3Ј-P site block 3Ј-P

Summary

Interactions between the backbone of the viral DNA and

Integrase Accesses Supple DNA Backbones from the Minor Groove the ST site were important within the target DNA [14]. Together, these observations suggest many stabilizing contacts between integrase, the tip of the viral cDNA. The use of synthetic oligonucleotides allows studies of the effects on integrase activity of site- placed DNA modifications. We examined the effect of several DNA backbone modifications on integrase activity. The effects of anomeric inversion on inteconserved adenine, as well as the two phosphates on the grase 3Ј-P and ST were examined using oligonucleotides concomplementary strand that are closest to the cleavage site, taining ␣-anomers around the 3Ј-P site (Fig. 2A). The two phosphates at and following of an ␣-anomer results in a change in the normal 5Ј–3Ј direc-

MATERIALS AND METHODS

RESULTS

Effect of Intercalating dA Adducts