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

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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-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

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Electron microscopy of the nucleoproteins of influenza A and B viruses in the absence or presence of different RNA and different buffers. The A/NP or B/NP were incubated overnight at room temperature with or without 5′phosphate-polyUC-FAM3′ in 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol (β-ME), and different NaCl concentrations from 50 to 300 mM. Samples were negatively stained with sodium silicotungstate. Electron micrographs show different oligomeric states depending on the salt concentration and the RNA used. The scale bar corresponds to 100 nm.
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viruses-08-00247-f004: Electron microscopy of the nucleoproteins of influenza A and B viruses in the absence or presence of different RNA and different buffers. The A/NP or B/NP were incubated overnight at room temperature with or without 5′phosphate-polyUC-FAM3′ in 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol (β-ME), and different NaCl concentrations from 50 to 300 mM. Samples were negatively stained with sodium silicotungstate. Electron micrographs show different oligomeric states depending on the salt concentration and the RNA used. The scale bar corresponds to 100 nm.

Mentions: In order to assess the influence of both salt and RNA size on the binding of monomeric NP, a series of salt conditions (50, 150 and 300 mM NaCl) were tested against size-incremented polyUC RNA from 5 to 24 nt. The experiments were performed using SEC-MALLS (Figure 3 and Figure S2a,b), negative staining EM (Figure 4 and Figure 5) and fluorescence anisotropy (Figure 6 and Figure S4). A 5′phosphate-polyUC-OH3′ was used for the MALLS experiments and 5′phosphate-polyUC-FAM3′ for the negative staining EM and for the fluorescence anisotropy experiments.


Binding of RNA by the Nucleoproteins of Influenza Viruses A and B
Electron microscopy of the nucleoproteins of influenza A and B viruses in the absence or presence of different RNA and different buffers. The A/NP or B/NP were incubated overnight at room temperature with or without 5′phosphate-polyUC-FAM3′ in 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol (β-ME), and different NaCl concentrations from 50 to 300 mM. Samples were negatively stained with sodium silicotungstate. Electron micrographs show different oligomeric states depending on the salt concentration and the RNA used. The scale bar corresponds to 100 nm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035961&req=5

viruses-08-00247-f004: Electron microscopy of the nucleoproteins of influenza A and B viruses in the absence or presence of different RNA and different buffers. The A/NP or B/NP were incubated overnight at room temperature with or without 5′phosphate-polyUC-FAM3′ in 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol (β-ME), and different NaCl concentrations from 50 to 300 mM. Samples were negatively stained with sodium silicotungstate. Electron micrographs show different oligomeric states depending on the salt concentration and the RNA used. The scale bar corresponds to 100 nm.
Mentions: In order to assess the influence of both salt and RNA size on the binding of monomeric NP, a series of salt conditions (50, 150 and 300 mM NaCl) were tested against size-incremented polyUC RNA from 5 to 24 nt. The experiments were performed using SEC-MALLS (Figure 3 and Figure S2a,b), negative staining EM (Figure 4 and Figure 5) and fluorescence anisotropy (Figure 6 and Figure S4). A 5′phosphate-polyUC-OH3′ was used for the MALLS experiments and 5′phosphate-polyUC-FAM3′ for the negative staining EM and for the fluorescence anisotropy experiments.

View Article: PubMed Central - PubMed

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-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

No MeSH data available.


Related in: MedlinePlus