title: Selection and analysis of HIV-1 integrase strand transfer inhibitor resistant mutant viruses.
abstract: This report describes methods for the selection and analysis of antiretroviral resistance to HIV integrase strand transfer inhibitors (InSTIs) in cell culture. The method involves the serial passage of HIV-1 in the presence of increasing concentrations of test inhibitors, followed by the cloning and sequencing of the integrase coding region from the selected viruses. The identified mutations are subsequently re-engineered into a reference wild-type molecular clone, and the resulting replication capacity and level of drug resistance are determined relative to the wild-type virus. Here we describe examples of selection and analysis of InSTI-resistant viruses using four integrase inhibitors from three structurally distinct chemical classes; a diketo acid, two naphthyridines, and a pyrimidinecarboxamide. Each inhibitor selected an independent route to resistance. Interestingly, the shift in the IC50 required to suppress the re-engineered resistant mutant viruses closely matched the concentration of compound used during the selection of drug resistance.
authors.affiliation: Department of Antiviral Research, Merck Research Laboratories, 770 Sumneytown Pike, WP26A-3000, West Point, PA 19486, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Witmer
authors.forename: Robert
authors.initials: R
authors.lastname: Danovich
mesh_terms: D002478:Cells, Cultured; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D019427:HIV Integrase / Q000235:genetics*; D019428:HIV Integrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000187:drug effects / Q000235:genetics*; D009287:Naphthyridines / Q000737:chemistry / Q000494:pharmacology; D016662:Virus Integration / Q000187:drug effects / Q000235:genetics*
chemical_list: D019428:HIV Integrase Inhibitors; C489415:L870810; D009287:Naphthyridines; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1