Binding of RNA by the Nucleoproteins of Influenza Viruses A and B

• Published in: Viruses Vol. 8; no. 9; p. 247
• Main Authors: Labaronne, Alice, Swale, Christopher, Monod, Alexandre, Schoehn, Guy, Crépin, Thibaut, Ruigrok, Rob W H
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 13.09.2016; MDPI AG
• Subjects: assembly; Binding Sites; Biochemistry, Molecular Biology; influenza virus; Life Sciences; nucleoprotein; oligomerization; Orthomyxoviridae - physiology; Protein Binding; ribonucleoprotein; RNA; RNA - metabolism; RNA-Binding Proteins - metabolism; Structural Biology; Viral Core Proteins - metabolism; Virus Assembly; assembly; influenza virus; nucleoprotein; RNA; oligomerization; ribonucleoprotein
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v8090247

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.