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HIV Protocols pp 119-131 | Cite as

Analysis of HIV-1 Gag-RNA Interactions in Cells and Virions by CLIP-seq

  • Sebla B. KutluayEmail author
  • Paul D. BieniaszEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1354)

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.

Key words

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

Notes

Acknowledgements

This work was supported by NIH grants R01AI501111 and P50GM103297. S.B.K. was supported in part by an AmFAR Mathilde Krim Postdoctoral Fellowship.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratory of Retrovirology, Aaron Diamond AIDS Research CenterThe Rockefeller UniversityNew YorkUSA
  2. 2.Department of Molecular MicrobiologyWashington University School of MedicineSt. LouisUSA
  3. 3.Howard Hughes Medical Institute, Aaron Diamond AIDS Research CenterThe Rockefeller UniversityNew YorkUSA

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