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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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X-ray structures of influenza virus nucleoproteins. (a) Influenza A nucleoprotein (A/NP) (PDB ID: 2IQH) in blue forms a trimer, whereas (b) Influenza B nucleoprotein (B/NP) (PDB ID: 3TJ0) in green forms a tetramer. The oligomerization loops are coloured red on both (a) and (b). (c) Overlay of A/NP and B/NP. The overall folds of A/NP and B/NP are very similar with a head and a body domain. The root-mean-square deviation (r.m.s.d.) is 1.49 Å for 458 Cα. The exchange domains of the two structures point in different directions because they are bound to different neighbours.
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viruses-08-00247-f001: X-ray structures of influenza virus nucleoproteins. (a) Influenza A nucleoprotein (A/NP) (PDB ID: 2IQH) in blue forms a trimer, whereas (b) Influenza B nucleoprotein (B/NP) (PDB ID: 3TJ0) in green forms a tetramer. The oligomerization loops are coloured red on both (a) and (b). (c) Overlay of A/NP and B/NP. The overall folds of A/NP and B/NP are very similar with a head and a body domain. The root-mean-square deviation (r.m.s.d.) is 1.49 Å for 458 Cα. The exchange domains of the two structures point in different directions because they are bound to different neighbours.

Mentions: For influenza virus, two structures of the nucleoprotein of influenza A (A/NP) and influenza B (B/NP) have been obtained: A/NP trimers [25,27] and its R416A monomeric mutant [28], and B/NP tetramers [24]. Figure 1 shows the structures of the A/NP trimer and B/NP tetramer. The way that this protein makes oligomers is through an oligomerization loop that goes from one protomer into its neighbour. The loop makes an angle from their head/body core, thus making either trimers or tetramers (Figure 1). Negative staining electron microscopy of NP from influenza A/PR/8/34 showed monomers and trimers and many larger oligomers, but no dimers [29]. Within this paper, we will show that B/NP can make dimers, probably because the angle of the loop allows dimers in contrast to A/NP. No structure of nucleoprotein–RNA complex exists for influenza virus. Early work using biochemistry and electron microscopy on RNPs showed that each protomer of nucleoprotein binds about 20 bases [30,31,32,33]. Later, Ortega et al. [34] reconstituted RNPs from NP, viral RNA polymerase, and several vRNAs with different sizes. It was found that the RNPs with 48 bases per pair of NPs amplify best. Hutchinson et al. [35] used mass spectrometry on purified A/WSN/33 virions and found that NP binds 26 bases (13,588 bases for 530 copies of NP per virion). However, the work on the nucleoprotein of infectious salmon anaemia virus (ISAV), a fish orthomyxovirus [36], suggested that NP only binds 12 bases, although the sizes of the RNPs are the same when compared to influenza A and B viruses. These authors suggested that only 12 bases bind in the potential RNA-binding groove, whereas the other 12 bases bind in another way to the nucleoprotein.


Binding of RNA by the Nucleoproteins of Influenza Viruses A and B
X-ray structures of influenza virus nucleoproteins. (a) Influenza A nucleoprotein (A/NP) (PDB ID: 2IQH) in blue forms a trimer, whereas (b) Influenza B nucleoprotein (B/NP) (PDB ID: 3TJ0) in green forms a tetramer. The oligomerization loops are coloured red on both (a) and (b). (c) Overlay of A/NP and B/NP. The overall folds of A/NP and B/NP are very similar with a head and a body domain. The root-mean-square deviation (r.m.s.d.) is 1.49 Å for 458 Cα. The exchange domains of the two structures point in different directions because they are bound to different neighbours.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-08-00247-f001: X-ray structures of influenza virus nucleoproteins. (a) Influenza A nucleoprotein (A/NP) (PDB ID: 2IQH) in blue forms a trimer, whereas (b) Influenza B nucleoprotein (B/NP) (PDB ID: 3TJ0) in green forms a tetramer. The oligomerization loops are coloured red on both (a) and (b). (c) Overlay of A/NP and B/NP. The overall folds of A/NP and B/NP are very similar with a head and a body domain. The root-mean-square deviation (r.m.s.d.) is 1.49 Å for 458 Cα. The exchange domains of the two structures point in different directions because they are bound to different neighbours.
Mentions: For influenza virus, two structures of the nucleoprotein of influenza A (A/NP) and influenza B (B/NP) have been obtained: A/NP trimers [25,27] and its R416A monomeric mutant [28], and B/NP tetramers [24]. Figure 1 shows the structures of the A/NP trimer and B/NP tetramer. The way that this protein makes oligomers is through an oligomerization loop that goes from one protomer into its neighbour. The loop makes an angle from their head/body core, thus making either trimers or tetramers (Figure 1). Negative staining electron microscopy of NP from influenza A/PR/8/34 showed monomers and trimers and many larger oligomers, but no dimers [29]. Within this paper, we will show that B/NP can make dimers, probably because the angle of the loop allows dimers in contrast to A/NP. No structure of nucleoprotein–RNA complex exists for influenza virus. Early work using biochemistry and electron microscopy on RNPs showed that each protomer of nucleoprotein binds about 20 bases [30,31,32,33]. Later, Ortega et al. [34] reconstituted RNPs from NP, viral RNA polymerase, and several vRNAs with different sizes. It was found that the RNPs with 48 bases per pair of NPs amplify best. Hutchinson et al. [35] used mass spectrometry on purified A/WSN/33 virions and found that NP binds 26 bases (13,588 bases for 530 copies of NP per virion). However, the work on the nucleoprotein of infectious salmon anaemia virus (ISAV), a fish orthomyxovirus [36], suggested that NP only binds 12 bases, although the sizes of the RNPs are the same when compared to influenza A and B viruses. These authors suggested that only 12 bases bind in the potential RNA-binding groove, whereas the other 12 bases bind in another way to the nucleoprotein.

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