Protein–Protein and Protein–RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding to HIV-1 Integrase

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages85-97
Number of pages13
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2610
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Alpha assay
  • HIV-1 IN
  • INI1/SMARCB1
  • Protien-protein interaction
  • Protien-RNA interaction
  • RNA mimicry
  • TAR RNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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