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

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.

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Homogeneous sample analysis of nucleoproteins of influenza A and B viruses. (a) Schematic representations of A/NP (blue) and B/NP (green). The A/NP presents a short 21 amino acid N-terminus, whereas B/NP has a longer N-tail of 72 amino acids; (b) Comparison of the size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis of A/NP and B/NP at 50 mM NaCl. Experimental molecular weights, next to the peaks, are coherent with the expected masses of 58 and 65 kDa for A/NP and B/NP, respectively; (c) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the purified A/NP and B/NP; Panels (d) and (e) are, respectively, the ultraviolet (UV) spectra of the A/NP and B/NP. Mw: molecular weight; Abs: absorbance.
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viruses-08-00247-f002: Homogeneous sample analysis of nucleoproteins of influenza A and B viruses. (a) Schematic representations of A/NP (blue) and B/NP (green). The A/NP presents a short 21 amino acid N-terminus, whereas B/NP has a longer N-tail of 72 amino acids; (b) Comparison of the size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis of A/NP and B/NP at 50 mM NaCl. Experimental molecular weights, next to the peaks, are coherent with the expected masses of 58 and 65 kDa for A/NP and B/NP, respectively; (c) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the purified A/NP and B/NP; Panels (d) and (e) are, respectively, the ultraviolet (UV) spectra of the A/NP and B/NP. Mw: molecular weight; Abs: absorbance.

Mentions: The nucleoproteins of influenza viruses A and B were purified as monomers with only 5% of the protein forming larger oligomers (Figure 2b). The UV spectra of each sample showed no nucleic acid contamination. The B/NP is longer than A/NP because of a longer N-terminal tail (Figure 2a and Figure S1).


Binding of RNA by the Nucleoproteins of Influenza Viruses A and B
Homogeneous sample analysis of nucleoproteins of influenza A and B viruses. (a) Schematic representations of A/NP (blue) and B/NP (green). The A/NP presents a short 21 amino acid N-terminus, whereas B/NP has a longer N-tail of 72 amino acids; (b) Comparison of the size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis of A/NP and B/NP at 50 mM NaCl. Experimental molecular weights, next to the peaks, are coherent with the expected masses of 58 and 65 kDa for A/NP and B/NP, respectively; (c) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the purified A/NP and B/NP; Panels (d) and (e) are, respectively, the ultraviolet (UV) spectra of the A/NP and B/NP. Mw: molecular weight; Abs: absorbance.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-08-00247-f002: Homogeneous sample analysis of nucleoproteins of influenza A and B viruses. (a) Schematic representations of A/NP (blue) and B/NP (green). The A/NP presents a short 21 amino acid N-terminus, whereas B/NP has a longer N-tail of 72 amino acids; (b) Comparison of the size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis of A/NP and B/NP at 50 mM NaCl. Experimental molecular weights, next to the peaks, are coherent with the expected masses of 58 and 65 kDa for A/NP and B/NP, respectively; (c) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the purified A/NP and B/NP; Panels (d) and (e) are, respectively, the ultraviolet (UV) spectra of the A/NP and B/NP. Mw: molecular weight; Abs: absorbance.
Mentions: The nucleoproteins of influenza viruses A and B were purified as monomers with only 5% of the protein forming larger oligomers (Figure 2b). The UV spectra of each sample showed no nucleic acid contamination. The B/NP is longer than A/NP because of a longer N-terminal tail (Figure 2a and Figure S1).

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