Analysis of HIV-1 Gag-RNA interactions in cells and virions by CLIP-seq

Sebla B. Kutluay, Paul D. Bieniasz

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Scopus citations

Abstract

Next-generation sequencing-based methodologies have revolutionized the analysis of protein-nucleic acid complexes; yet these novel approaches have rarely been applied in virology. Because it has an RNA genome, RNA-protein interactions play critical roles in human immunodeficiency virus type 1 (HIV-1) replication. In many cases, the binding sites of proteins on HIV-1 RNA molecules in physiologically relevant settings are not known. Cross-linking-immunoprecipitation sequencing (CLIP-seq) methodologies, which combine immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, is a powerful technique that can be applied to such questions as it provides a global account of RNA sequences bound by a RNA-binding protein of interest in physiological settings at near-nucleotide resolution. Here, we describe the application of the CLIP-seq methodology to identify the RNA molecules that are bound by the HIV-1 Gag protein in cells and in virions. This protocol can easily be applied to other viral and cellular RNA-binding proteins that influence HIV-1 replication.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages119-131
Number of pages13
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1354
ISSN (Print)1064-3745

Keywords

  • Bioinformatics
  • CLIP-seq
  • Cells
  • Gag
  • HIV-1
  • Next-generation sequencing
  • Protein-RNA interaction
  • RNA packaging
  • RNA-binding protein
  • UV cross-linking
  • Virions

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