title: Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase.
abstract: HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles.
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA. kessl.1@osu.edu.
authors.forename: Jacques J
authors.initials: JJ
authors.lastname: Kessl
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.|Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D000067493:Dynamic Light Scattering / Q000379:methods; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D019427:HIV Integrase / Q000737:chemistry / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008826:Microbial Sensitivity Tests / Q000379:methods; D055503:Protein Multimerization / Q000187:drug effects*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014771:Virion / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D019428:HIV Integrase Inhibitors; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1
keywords: Chemical cross-linking; Dynamic light scattering; HIV-1 integrase; Homogenous time-resolved fluorescence; Protein multimerization