title: Identification of RNA partners of viral proteins in infected cells.
abstract: RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5' copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.
authors.affiliation: Unité de Génomique virale et Vaccination, Institut Pasteur, CNRS URA-3015, Paris, France.
authors.forename: Anastassia V
authors.initials: AV
authors.lastname: Komarova
authors.forename: Chantal
authors.initials: C
authors.lastname: Combredet
authors.forename: Odile
authors.initials: O
authors.lastname: Sismeiro
authors.forename: Marie-Agnès
authors.initials: MA
authors.lastname: Dillies
authors.forename: Bernd
authors.initials: B
authors.lastname: Jagla
authors.forename: Raul Yusef
authors.initials: RY
authors.lastname: Sanchez David
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vabret
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Coppée
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Vidalain
authors.forename: Frédéric
authors.initials: F
authors.lastname: Tangy
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008459:Measles virus / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011971:Receptors, Immunologic; D011994:Recombinant Proteins / Q000378:metabolism; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases
keywords: RNA-protein interactions; affinity purification; measles virus; next-generation sequencing; nucleocapsids; tagged proteins