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Viruses. 2016 Sep 13;8(9). pii: E247. doi: 10.3390/v8090247.

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

Author information

1
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. alice.tissot@ibs.fr.
2
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. cswale@embl.fr.
3
EMBL Grenoble Outstation, 71 Avenue des Martyrs, BP181, F-38042 Grenoble CEDEX 9, France. cswale@embl.fr.
4
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. alexandre.monod@orange.fr.
5
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. guy.schoehn@ibs.fr.
6
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. thibaut.crepin@ibs.fr.
7
Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France. rob.ruigrok@ibs.fr.

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.

KEYWORDS:

RNA; assembly; influenza virus; nucleoprotein; oligomerization; ribonucleoprotein

PMID:
27649229
PMCID:
PMC5035961
DOI:
10.3390/v8090247
[Indexed for MEDLINE]
Free PMC Article

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