title: Biochemical analysis of HIV-1 integrase variants resistant to strand transfer inhibitors.
abstract: 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.
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA. Ira.Dicker@BMS.com
authors.forename: Ira B
authors.initials: IB
authors.lastname: Dicker
authors.forename: Brian
authors.initials: B
authors.lastname: Terry
authors.forename: Zeyu
authors.initials: Z
authors.lastname: Lin
authors.forename: Zhufang
authors.initials: Z
authors.lastname: Li
authors.forename: Sagarika
authors.initials: S
authors.lastname: Bollini
authors.forename: Himadri K
authors.initials: HK
authors.lastname: Samanta
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Gali
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walker
authors.forename: Mark R
authors.initials: MR
authors.lastname: Krystal
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D002460:Cell Line; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000187:drug effects* / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019428:HIV Integrase Inhibitors / Q000737:chemistry*; D016325:HIV Long Terminal Repeat / Q000502:physiology; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D016662:Virus Integration / Q000187:drug effects / Q000502:physiology
chemical_list: D004279:DNA, Viral; D019428:HIV Integrase Inhibitors; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1