The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein

Abstract

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.

Figure 1. Gel filtration chromatograms for ISAV-NP proteins.

The eluted positions of three protein standards are marked by arrows.

Figure 2. ISAV-NP crystal structure.

(A) Three orthogonal views of an NP dimer. One subunit is colored in green, whereas the other subunit is colored by domains with blue for the N-terminal domain, yellow for the head, red for the body, and magenta for the tail loop. In the last panel, the magenta tail loop is shown by space-filling model to highlight the extensive interaction mediated by the tail loop. (B) ISAV-NP (red) superimposed onto the influenza A virus NP (cyan, PDB ID: 2IQH) . (C) ISAV-NP monomer. The molecule is colored continuously from blue to red for the N-terminus to C-terminus, respectively. Secondary structural elements are numbered. (D) Calculated electronic potential for an NP monomer. Positively charged residues are highlighted. (B–D) are shown in the same orientation by viewing into the putative RNA binding groove, same as the middle panel in (A). (E) ISAV-NP secondary structure assignment. α-helices are shown by cylinders and the β-strands are represented by arrows. Conserved charged residues from the RNA binding groove are highlighted in blue. The conserved aromatic residue F274 in the groove is shown in red. The NP sequences from ISAV and the influenza A virus were manually aligned, based on tertiary structures.

Figure 3. ISAV-NP RNA binding.

(A–C) RNA binding affinity measurements for the wt NP (A), ΔN111 (B) and ΔC16 (C). FA was performed using three RNA oligos each containing 20, 24, and 28 nts. The binding curves are plotted using the same protein concentration range. For the ΔC16 mutant, an inset is added in (C) to provide a more spread-out view at lower protein concentrations. Fully saturated curves for the 20-nt RNA are shown in Fig. S3 in Supporting Information. (D–G) RNA binding stoichiometry measurements for the wt NP and the ΔLOOP monomeric mutant. NP:RNA complexes assembled in each experiments are schematically shown with blue handcuffs for NP dimers and grey curves for RNA molecules.

Figure 4. ISAV-NP:RNA complexes.

(A–F) EM images of the free NP (A, B), NP bound to a 24-nt RNA (C, D), and NP bound to a 48-nt RNA (E, F). (G) Molecular weights of NP and NP:RNA complexes as determined by DLS. Molecular interpretations were made for each figure with blue handcuffs for the NP dimers and grey curves for RNA molecules.