title: Binding of RNA by the Nucleoproteins of Influenza Viruses A and B.
abstract: This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)₆-FAM(3') RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. alice.tissot@ibs.fr.
authors.forename: Alice
authors.initials: A
authors.lastname: Labaronne
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. cswale@embl.fr.|EMBL Grenoble Outstation, 71 Avenue des Martyrs, BP181, F-38042 Grenoble CEDEX 9, France. cswale@embl.fr.
authors.forename: Christopher
authors.initials: C
authors.lastname: Swale
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. alexandre.monod@orange.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Monod
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. guy.schoehn@ibs.fr.
authors.forename: Guy
authors.initials: G
authors.lastname: Schoehn
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. thibaut.crepin@ibs.fr.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Crépin
authors.affiliation: Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. rob.ruigrok@ibs.fr.
authors.forename: Rob W H
authors.initials: RW
authors.lastname: Ruigrok
mesh_terms: D001665:Binding Sites; D019590:Nucleocapsid Proteins; D009975:Orthomyxoviridae / Q000502:physiology*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; C588186:nucleoprotein, influenza B virus; D012313:RNA
keywords: RNA; assembly; influenza virus; nucleoprotein; oligomerization; ribonucleoprotein