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Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone

Nature Structural & Molecular Biology volume 21, pages 754–759 (2014)Cite this article

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Abstract

Nipah virus (NiV) is a highly pathogenic emergent paramyxovirus causing deadly encephalitis in humans. Its replication requires a constant supply of unassembled nucleoprotein (N0) in complex with its viral chaperone, the phosphoprotein (P). To elucidate the chaperone function of P, we reconstituted NiV the N0–P core complex and determined its crystal structure. The binding of the N-terminal region of P blocks the polymerization of N by interfering with subdomain exchange between N protomers and keeps N0 in an open conformation, ready to grasp an RNA molecule. We found that a peptide derived from the N-binding region of P protects cells against viral infection and demonstrated by structure-based mutagenesis that this peptide acts by inhibiting N0–P formation. These results provide new insights about the assembly of N along genomic RNA and validate the N0–P complex as a target for drug development.

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Figure 1: Structure of reconstituted NiV N0–P complex in solution and in crystal.
Figure 2: Comparison of NiV and RSV N proteins reveals an open-to-closed conformational change.
Figure 3: Conservation of the N0-P interface and inhibition of NiV replication by the N0-binding peptide of P.
Figure 4: Chaperone activities of NiV P.
Figure 5: Proposed mechanism for RNA encapsidation in the Paramyxovirinae subfamily.

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Acknowledgements

We thank W. Burmeister and A. McCarthy for their help with X-ray data collection and C. Leyrat for discussions. F.Y. was supported by a predoctoral fellowship from the Région Rhône-Alpes. This work was supported by grants from the French Agence Nationale de la Recherche to M.J. (ANR-07-001-01) and to V.V. (ANR-09-MIEN-018-01), from the European Commission's FP7 program ANTIGONE (278976) to V.V. and from the Fondation Innovations en Infectiologie (FINOVI) to V.V. and M.J. This work used the platforms of the Grenoble Instruct center (Integrated Structural Biology Grenoble; UMS3518 CNRS-CEA-UJF-EMBL) with support from The French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INSB-05-02) and The Alliance Grenobloise pour la Biologie Structurale et Cellulaire Intégrées (GRAL) (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).

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Authors and Affiliations

  1. Université Grenoble Alpes, Unit of Virus Host Cell Interactions, Grenoble, France

    Filip Yabukarski, Nicolas Tarbouriech, Jean-Marie Bourhis, Rob W H Ruigrok & Marc Jamin

  2. CNRS, Unit of Virus Host Cell Interactions, Grenoble, France

    Filip Yabukarski, Nicolas Tarbouriech, Jean-Marie Bourhis, Rob W H Ruigrok & Marc Jamin

  3. International Centre for Research in Infectiology (CIRI), INSERM U1111–CNRS UMR5308, Université Lyon 1, Ecole Normale Supérieure de Lyon, Lyon, France

    Philip Lawrence & Viktor Volchkov

  4. Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France

    Elise Delaforge, Malene Ringkjøbing Jensen & Martin Blackledge

  5. CNRS, Institut de Biologie Structurale, Grenoble, France

    Elise Delaforge, Malene Ringkjøbing Jensen & Martin Blackledge

  6. Commissariat à l′Énergie Atomique (CEA), Institut de Biologie Structurale, Grenoble, France

    Elise Delaforge, Malene Ringkjøbing Jensen & Martin Blackledge

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  1. Filip Yabukarski
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Contributions

F.Y., P.L., M.R.J., R.W.H.R., M.B., V.V. and M.J. designed all experiments. F.Y., P.L., E.D. and M.R.J. performed the experiments. P.L. performed BSL-4 experiments. F.Y., P.L., N.T., J.-M.B., M.R.J., R.W.H.R., M.B., V.V. and M.J. contributed to data analysis. F.Y., P.L., M.R.J., M.B., V.V. and M.J. wrote the paper.

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Correspondence to Viktor Volchkov or Marc Jamin.

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Integrated supplementary information

Supplementary Figure 1 Structural characterization of NiV N0–P in solution and in crystal.

(a) SAXS analysis of the N32-3830-P50 complex. The Guinier plot for complex concentrations of 0.55, 1.1, 1.6 and 2.4 mg.mL-1 shows no evidence for aggregation or intermolecular interactions. The averaged intensity at zero angle corrected for protein concentration (I(0)/C) value of 46 ± 1 kDa is in good agreement with the theoretical molecular mass of the heterodimeric complex (45,613 Da). (b) Superposition of the experimental SAXS curve in black (2.4 mg.mL-1) with the theoretical curve (in red) back calculated for the averaged ab initio bead model (20 independent models; normalized spatial discrepancy (NSD) < 1.0) generated with DAMMIN28 and DAMAVER29. (c) The heterodimeric N32-3830-P50 complex has a bean shape in solution. The ab initio bead model generated from SAXS data (in grey) accommodates a single N32-3830-P50 copy from the crystal. (d) Comparison of the 2D 1H-15N heteronuclear single-quantum coherence (HSQC) NMR spectra of free P100 (in red) and of P100 bound to N32-402(in blue). (e) Electrostatic surface potential of NiV and RSV N (PDB code 2WJ8; ref. 5) proteins at ± 5 kTe-1: blue (basic), white (neutral) and red (acidic). The hypothetical RNA binding site is indicated in NiV N, and bound RNA in the RSV complex is shown in orange. (f) Western blot using in-house henipavirus specific rabbit anti-N antibody with cell lysates from Nipah infected Vero E6 cells lysed 48h post-infection (NiV, left lane) compared to uninfected cells (NI, right lane). Theoretical molecular mass of the NiV N protein is 58,168 Da.

Supplementary Figure 2 NiV P100 is globally disordered in solution but contains fluctuating α-helical elements.

(a,b). Molecular size. The hydrodynamic radius of 2.6 ± 0.5 nm measured by SEC (a) and the radius of gyration of 3.1 ± 0.5 nm measured by SAXS (b) are larger than expected for a compact domain of this size. SAXS profiles were recorded at 0.3, 0.5 and 0.6 mg.mL-1. (c) NMR spectroscopy. The poor chemical shift dispersion of amide 1H resonances in the heteronuclear single quantum coherence (HSQC) NMR spectrum (Supplementary Fig. 1d) is typical of disordered proteins. The secondary structure propensity (SSP) parameter calculated from Cα and Cβ secondary chemical shifts indicates the presence of fluctuating helices (red boxes). The position of helices in the N-terminal region of P in the crystal structure of the N32-3830-P50 complex is shown below. (d) The N0-binding region of Paramyxovirinae P contains two conserved motifs (Soyuz1 and Soyuz2)23. Conserved residues are colored according to their properties: acidic residues in violet, basic in red, hydrophobic in blue, polar in green and glycine in orange. G10 and I17 (larger letters) were mutated into arginine in order to destabilize the N0-P complex.

Supplementary Figure 3 The crystallographic asymmetric unit contains three copies of the N32–3830–P50 complex.

(a) Initial plate cluster used for microseeding. (b) Typical crystal obtained for the N32-3830-P50 from microseeding and used for data collection. (c) Overall view of crystal packing of the N32-3830-P50 complex. The three copies of the complex in the asymmetric unit are colored in blue and red. The packing shows no indication of specific oligomerization of N in the crystal. (d,e). Experimental selenium SAD phased density map of P50 (d) and N0 (e) in the N32-3830-P50 complex. The final model after refinement is superposed on the map at contour level 1 σ. Only one N0-P copy from the asymmetric unit is represented. (f,g) Final 2Fo-Fc difference electron density map of P50 (f) and N0 (g) in the N32-3830-P50 complex contoured at 1 σ. (h). Comparison of the different copies of the N32-3830-P50 complex present in the asymmetric unit. Two copies are well defined in the SAD phased map, whereas NNTD of the third one is less densely packed in the crystal and the corresponding electron density is ill-defined. (i) Structural overlay of the three copies of N. Slightly different orientations of secondary structure elements in NNTD suggest that this region of the protein is dynamic, at least in the absence of RNA.

Supplementary Figure 4 Comparison of NNVs N 3D fold.

(a) Conserved architecture of the N proteins: NiV, Nipah virus (Paramyxoviridae - Paramyxovirinae); RSV, respiratory syncytial virus (Paramyxoviridae - Pneumovirinae) (PDB code 2WJ8; ref 5); BDV, Borna disease virus (Bornaviridae) (PDB code 1N93; ref 19); VSV, vesicular stomatitis virus (Rhabdoviridae) (PDB code 2GIC; ref 7). Pairwise structural alignments led us to define four subdomains in the N core, namely NNTD1, NNTD2, NNTD3 and NCTD. The structures are similarly oriented and colored according to subdomains. VSV N contains an additional C-terminal subdomain (NCTD2 in purple) comprising three α-helices, which form the surface of binding of the C-terminal domain of P. (b-d). Superpositions of subdomains from the different proteins. The NiV N subdomains are shown in the same colors as in panel a and the corresponding subdomains from the other viruses are shown in grey: NNTD1 (b), NNTD3 (c) and NCTD (d). NNV N proteins differ mainly in the relative orientations of these three domains and the structure of the variable NNTD2 region. (e) Pairwise comparison of NiV N and human RSV N. Structure-based sequence alignment of NiV N (PDB code 4CO6) and RSV N (PDB code 2WJ8; ref 5). Secondary structure elements and residue numbering of NiV N are indicated above and secondary structure elements of RSV are indicated below.

Supplementary Figure 5 RNA grasp and hinge motions.

(a) Normal mode analysis (NMA) and hypothetical hinge motion in NiV N. Elastic network NMA was carried out on the N0 molecule to explore its motions45. Representative models of the two lowest-frequency modes show motions of NNTD relative to NCTD with a hinge at the junction between the two domains (red circle) in agreement with a mechanism of closure of the RNA binding cavity. (b) The second lowest mode (mode 8) captures a rotation of NNTD, which agrees with the hypothetical closure of the protein around the RNA. The initial model (in grey) superposes with the crystal structure of NiV N0 in blue. (c) After displacement along mode 8, the model (in wheat) superposes with RSV N structure taken from the N-RNA complex (in light blue). (d) Close-up of RSV N-RNA complex showing that helices αN6a and αN9 pack against the RNA molecule. G241 and G245 are shown in yellow. (e) Close-up of NiV N in the N0-P crystal structure (left panel) and in a hypothetical closed conformation (right panel). The RNA molecule (in grey) is positioned against NCTD as in RSV NC. Several residues of conserved motif 3 and conserved D254 interfere with base 1 binding in the closed form.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 4733 kb)

Proposed mechanism of conformational change in NiV N upon RNA binding.

The animation shows the proposed hinge motion of N lobes upon RNA binding with a pivot point at the junction of NNTD and NCTD. The RNA molecule is positioned against NCTD as in RSV N-RNA complex, but only 6 nt are shown in accordance with the rule of six enforced in the subfamily Paramyxovirinae. (WMV 48531 kb)

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Yabukarski, F., Lawrence, P., Tarbouriech, N. et al. Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone. Nat Struct Mol Biol 21, 754–759 (2014). https://doi.org/10.1038/nsmb.2868

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