Kessl Jacques J, Sharma Amit, Kvaratskhelia Mamuka
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.
Methods Mol Biol. 2016;1354:149-64. doi: 10.1007/978-1-4939-3046-3_10.
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.
HIV-1整合酶（IN）是一个重要的治疗靶点，因为其功能对于病毒生命周期至关重要。多功能变构IN抑制剂（ALLINIs）的发现，通过促进异常的高阶IN多聚化以及抑制IN与其细胞辅因子LEDGF/p75的相互作用，有效地损害病毒复制，为将IN多聚化作为治疗靶点开辟了新途径。此外，最近发现的多聚化选择性IN抑制剂（MINIs），为研究体外和感染细胞中异常IN多聚化的直接影响提供了新的研究探针。在此，我们描述了三种互补方法，旨在检测和量化这些新型抑制剂对IN多聚化的影响。这些方法包括基于均相时间分辨荧光的测定法，可测量抑制剂诱导的异常IN多聚化的半数有效浓度（EC50）值；基于动态光散射的测定法，可随时间监测寡聚IN颗粒的形成和大小；以及基于化学交联的相互作用IN亚基测定法，可确定病毒颗粒中的IN寡聚体。
