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J. Biol. Chem., Vol. 282, Issue 43, 31186-31196, October 26, 2007

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Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors^*

Ira B. Dicker¹, Himadri K. Samanta, Zhufang Li, Yang Hong, Yuan Tian, Jacques Banville^¶, Roger R. Remillard^¶, Michael A. Walker^¶, David R. Langley^||, and Mark Krystal

From the Departments of Virology, ^¶Medicinal Chemistry, and ^||Computer Assisted Drug Design, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492 and the Radiochemistry Group, Department of Chemical Synthesis, Pharmaceutical Research Institute, Princeton, New Jersey 08540

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G²T¹C^–1A^–2) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G²:C² base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln¹⁴⁸ interacts with G², T¹, and C^–1 at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Received for publication, June 14, 2007 , and in revised form, August 20, 2007.

^* 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: Bristol-Myers Squibb Co., 5 Research Pkwy., Wallingford, CT 06492. Tel.: 203-677-7736; Fax: 203-677-6088; E-mail: Ira.Dicker{at}BMS.com.