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Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase

  • Protocol
HIV Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1354))

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.

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Acknowledgments

We thank Dr. Ross C. Larue for critical reading of the manuscript and valuable suggestions. The present study was supported by the National Institutes of Health (grants AI110270 to J.J.K., AI062520 and AI110310 to M.K.).

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Authors and Affiliations

  1. Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA

    Jacques J. Kessl, Amit Sharma & Mamuka Kvaratskhelia

  2. Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA

    Amit Sharma

Authors
  1. Jacques J. Kessl
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  2. Amit Sharma
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  3. Mamuka Kvaratskhelia
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Correspondence to Jacques J. Kessl .

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Editors and Affiliations

  1. Department of Microbiology & Immunology, Albert Einstein College of Medicine, BRONX, New York, USA

    Vinayaka R. Prasad

  2. Department of Genetics, Albert Einstein College of Medicine, BRONX, New York, USA

    Ganjam V. Kalpana

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Kessl, J.J., Sharma, A., Kvaratskhelia, M. (2016). Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_10

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  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_10

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

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