Limits...
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.

No MeSH data available.


Related in: MedlinePlus

SEC-MALLS analysis of oligomeric states of the nucleoproteins of influenza A and B virus according to RNA length. SEC-MALLS analysis were performed on a Superdex 200 increase column equilibrated with 20 mM Tris-HCl pH 7.5 and different NaCl concentrations, from 50 to 300 mM. Protein is injected alone or in complex (ratio 1:1) with a short 5′phosphate-polyUC-OH3′ RNA from five to 24 nucleotides. Complexes are incubated 1 h at room temperature before injection. For each buffer condition, the relative proportion of oligomers eluted from the column gradually increased upon increasing RNA length. Peaks are labelled 1 for monomer, 2 for dimer, 3 for trimer, 4 for tetramer, 5 for pentamer, and >6 for larger than hexamer.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5035961&req=5

viruses-08-00247-f003: SEC-MALLS analysis of oligomeric states of the nucleoproteins of influenza A and B virus according to RNA length. SEC-MALLS analysis were performed on a Superdex 200 increase column equilibrated with 20 mM Tris-HCl pH 7.5 and different NaCl concentrations, from 50 to 300 mM. Protein is injected alone or in complex (ratio 1:1) with a short 5′phosphate-polyUC-OH3′ RNA from five to 24 nucleotides. Complexes are incubated 1 h at room temperature before injection. For each buffer condition, the relative proportion of oligomers eluted from the column gradually increased upon increasing RNA length. Peaks are labelled 1 for monomer, 2 for dimer, 3 for trimer, 4 for tetramer, 5 for pentamer, and >6 for larger than hexamer.

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
SEC-MALLS analysis of oligomeric states of the nucleoproteins of influenza A and B virus according to RNA length. SEC-MALLS analysis were performed on a Superdex 200 increase column equilibrated with 20 mM Tris-HCl pH 7.5 and different NaCl concentrations, from 50 to 300 mM. Protein is injected alone or in complex (ratio 1:1) with a short 5′phosphate-polyUC-OH3′ RNA from five to 24 nucleotides. Complexes are incubated 1 h at room temperature before injection. For each buffer condition, the relative proportion of oligomers eluted from the column gradually increased upon increasing RNA length. Peaks are labelled 1 for monomer, 2 for dimer, 3 for trimer, 4 for tetramer, 5 for pentamer, and >6 for larger than hexamer.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-08-00247-f003: SEC-MALLS analysis of oligomeric states of the nucleoproteins of influenza A and B virus according to RNA length. SEC-MALLS analysis were performed on a Superdex 200 increase column equilibrated with 20 mM Tris-HCl pH 7.5 and different NaCl concentrations, from 50 to 300 mM. Protein is injected alone or in complex (ratio 1:1) with a short 5′phosphate-polyUC-OH3′ RNA from five to 24 nucleotides. Complexes are incubated 1 h at room temperature before injection. For each buffer condition, the relative proportion of oligomers eluted from the column gradually increased upon increasing RNA length. Peaks are labelled 1 for monomer, 2 for dimer, 3 for trimer, 4 for tetramer, 5 for pentamer, and >6 for larger than hexamer.
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