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

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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

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  • DOI: 10.1007/978-1-4939-3046-3_8
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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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Correspondence to Sebla B. Kutluay or Paul D. Bieniasz .

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Kutluay, S.B., Bieniasz, P.D. (2016). Analysis of HIV-1 Gag-RNA Interactions in Cells and Virions by CLIP-seq. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_8

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  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_8

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

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