title: Analysis of HIV-1 Gag-RNA Interactions in Cells and Virions by CLIP-seq.
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.
authors.affiliation: Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Avenue, New York, NY, 10016, USA. skutluay@wusm.wustl.edu.|Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO, 63110, USA. skutluay@wusm.wustl.edu.
authors.forename: Sebla B
authors.initials: SB
authors.lastname: Kutluay
authors.affiliation: Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Avenue, New York, NY, 10016, USA. pbienias@adarc.org.|Howard Hughes Medical Institute, Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Avenue, New York, NY, 10016, USA. pbienias@adarc.org.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Bieniasz
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015683:Gene Products, gag / Q000378:metabolism*; D023281:Genomics / Q000379:methods; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014466:Ultraviolet Rays; D014771:Virion / Q000235:genetics / Q000378:metabolism*
chemical_list: D015683:Gene Products, gag; D012367:RNA, Viral; D016601:RNA-Binding Proteins
keywords: Bioinformatics; CLIP-seq; Cells; Gag; HIV-1; Next-generation sequencing; Protein –RNA interaction; RNA -binding protein; RNA packaging; UV cross-linking; Virions