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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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Schematic representation of the oligomerization of influenza A and B viruses’ nucleoproteins depending on the size of the RNA molecules at 150 mM NaCl. Panels (a) and (b) are for A/NP and B/NP, respectively.
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viruses-08-00247-f007: Schematic representation of the oligomerization of influenza A and B viruses’ nucleoproteins depending on the size of the RNA molecules at 150 mM NaCl. Panels (a) and (b) are for A/NP and B/NP, respectively.

Mentions: At 50 mM NaCl we found that NP remains monomeric with small RNAs (up to 6 nt; Figure 7a). With 8 nt and longer RNA molecules, the monomers bind the RNA and then make trimers. At 150 mM NaCl, the monomers bind the RNA and then make trimers and larger complexes with 24-nt RNA. At 300 mM NaCl, part of the protein is already trimeric, so the binding of the RNA is to monomers and trimers and then all the complexes become trimers, making larger complexes with 24-nt RNA. The affinities at 50 mM NaCl are all similar, suggesting that the RNA is bound at the same site on the protein although 12-nt RNA binds slightly better than a 24-nt RNA (Figure 6). With more salt, shorter RNAs bind much less well but 24-nt RNA binds better than at 50 mM NaCl. At 300 mM NaCl, the trend is even stronger. Because the binding with a 24-nt RNA is stronger, this suggests that the RNA binding site is different for a 24-nt RNA at 150 and 300 mM NaCl, probably because under these conditions the RNA binds trimers and not monomers. Because the affinity of the RNA for the trimer is so strong, the trimers may have a problem disassembling and forming longer complexes.


Binding of RNA by the Nucleoproteins of Influenza Viruses A and B
Schematic representation of the oligomerization of influenza A and B viruses’ nucleoproteins depending on the size of the RNA molecules at 150 mM NaCl. Panels (a) and (b) are for A/NP and B/NP, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-08-00247-f007: Schematic representation of the oligomerization of influenza A and B viruses’ nucleoproteins depending on the size of the RNA molecules at 150 mM NaCl. Panels (a) and (b) are for A/NP and B/NP, respectively.
Mentions: At 50 mM NaCl we found that NP remains monomeric with small RNAs (up to 6 nt; Figure 7a). With 8 nt and longer RNA molecules, the monomers bind the RNA and then make trimers. At 150 mM NaCl, the monomers bind the RNA and then make trimers and larger complexes with 24-nt RNA. At 300 mM NaCl, part of the protein is already trimeric, so the binding of the RNA is to monomers and trimers and then all the complexes become trimers, making larger complexes with 24-nt RNA. The affinities at 50 mM NaCl are all similar, suggesting that the RNA is bound at the same site on the protein although 12-nt RNA binds slightly better than a 24-nt RNA (Figure 6). With more salt, shorter RNAs bind much less well but 24-nt RNA binds better than at 50 mM NaCl. At 300 mM NaCl, the trend is even stronger. Because the binding with a 24-nt RNA is stronger, this suggests that the RNA binding site is different for a 24-nt RNA at 150 and 300 mM NaCl, probably because under these conditions the RNA binds trimers and not monomers. Because the affinity of the RNA for the trimer is so strong, the trimers may have a problem disassembling and forming longer complexes.

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