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J. Biol. Chem., Vol. 283, Issue 35, 23599-23609, August 29, 2008

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Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors^*

Ira B. Dicker¹, Brian Terry, Zeyu Lin, Zhufang Li, Sagarika Bollini, Himadri K. Samanta, Volodymyr Gali, Michael A. Walker, and Mark R. Krystal

From the Departments of Virology and Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, Connecticut 06492

In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k_p/K_m) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k_p) values. However, smaller second-order effects were caused by up to 4-fold increases in K_m values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3'-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

Received for publication, June 2, 2008 , and in revised form, June 20, 2008.

^* 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.

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