title: Structure-analysis of the HIV-1 integrase Y143C/R raltegravir resistance mutation in association with the secondary mutation T97A.
abstract: The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study (S. Reigadas et al., PLoS One 5:e10311, 2010), we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing RAL-containing regimens. In these patients, the Y143C/R mutation was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro was to evaluate whether the secondary mutation, T97A, associated with the Y143C/R mutation could increase the level of resistance to RAL and impact IN activities. Site-directed mutagenesis experiments were performed with expression vectors harboring the region of the pol gene coding for IN. With a 3'-end processing assay, the 50% inhibitory concentrations (IC(50)) were 1.2 μM, 1.2 μM, 2.4 μM (fold change [FC], 2), and 20 μM (FC, 16.7) for IN wild type (WT), the IN T97A mutation, the IN Y143C/T97A mutation, and the IN Y143R/T97A mutation, respectively. FCs of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A mutations, with IC(50) of 0.625 μM and 2.5 μM, respectively. In the strand transfer assay, the IN Y143C or R mutation combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to results with the IN Y143C or R mutation alone. Assays without RAL suggested that the T97A mutation could rescue the catalytic activity which was impaired by the presence of the Y143C/R mutation. The combination of the T97A mutation with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This result indicates that the emergence of the Y143C/R/T97A double-mutation pattern in patients is a signature of a high resistance level.
authors.affiliation: UMR 5234, Laboratoire de Microbiologie Fondamentale et Pathogénicité, CNRS, Université Victor Segalen, Bordeaux, France. sandrine.reigadas@chu-bordeaux.fr
authors.forename: S
authors.initials: S
authors.lastname: Reigadas
authors.forename: B
authors.initials: B
authors.lastname: Masquelier
authors.forename: C
authors.initials: C
authors.lastname: Calmels
authors.forename: M
authors.initials: M
authors.lastname: Laguerre
authors.forename: E
authors.initials: E
authors.lastname: Lazaro
authors.forename: M
authors.initials: M
authors.lastname: Vandenhende
authors.forename: D
authors.initials: D
authors.lastname: Neau
authors.forename: H
authors.initials: H
authors.lastname: Fleury
authors.forename: M L
authors.initials: ML
authors.lastname: Andréola
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics*; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011760:Pyrrolidinones / Q000494:pharmacology*; D000068898:Raltegravir Potassium; D013329:Structure-Activity Relationship
chemical_list: D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1