Abstract
INI1/SMARCB1 is a host protein that interacts with HIV-1 integrase (IN) and influences multiple stages of viral replication. IN is a viral enzyme responsible for integration, and it also binds to HIV-1 genomic RNA. Recent studies from our laboratory demonstrated that IN-interacting Rpt1 (Repeat 1) domain of INI1 and TAR RNA region of HIV-1 genome both bind to the same residues and surface of IN C-terminal domain (CTD). Based on a series of analyses, we found that INI1-Rpt1 and TAR RNA structurally mimic each other and that IN mutants defective for binding to INI1 are also defective for binding to RNA and produce morphologically defective virions. The similarity of INI1-Rpt1 and TAR RNA in binding to IN was established by testing the binding of IN-CTD mutants with INI1-Rpt1 and TAR RNA using the Alpha assay. Here, I describe Alpha assay methods to compare the binding of INI1-Rpt1 protein and HIV-1 TAR RNA to IN-CTD and describe a three-component assay to demonstrate the competition between TAR RNA and INI1-Rpt1 to bind to IN.
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Acknowledgments
I thank Dr. Vinayaka Prasad and Dr. Rajiv Pathak for critically reading the manuscript. This study was supported by National Institute of Health GM112520-02, GM112520-04S1, and 1R01AI170206 to GVK.
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Kalpana, G.V. (2023). Protein–Protein and Protein–RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding to HIV-1 Integrase. In: Aquino de Muro, M. (eds) Virus-Host Interactions. Methods in Molecular Biology, vol 2610. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2895-9_8
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DOI: https://doi.org/10.1007/978-1-0716-2895-9_8
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