Analysis of HIV-1 Gag-RNA Interactions in Cells and Virions by CLIP-seq.
Methods in molecular biology, January 2016
Kutluay, Sebla B, Bieniasz, Paul D, Sebla B. Kutluay, Paul D. Bieniasz
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.
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