Structural insights into the Nipah virus nucleocapsid assembly

Figures

• 

  Fig. 1. Overall representation of NiV nucleocapsid assembly. A Schematic domain composition of the NiV N protein. The NT-arm, NT-Ncore, CT-Ncore, and CT-arm are colored in yellow, turquoise blue, pink, and frog green, respectively. B Cryo-EM map and model of the NiV nucleocapsid assembly. Domains are colored as described in panel A. C Surface representation of side and top projections of the pseudo-helical assembly. The indicated helical parameters (p, pitch; Δz, rise; Δφ, twist) are mean values derived from the 13 successive rigid-body transforms between adjacent N protomers (n → n + 1) in the C1 atomic model, with pitch computed from the mean rise and twist. Inner and outer diameters are also indicated. D Topology of the N protein. E Cartoon representation of the N protein monomer bound to ssRNA, fitted in the EM map. RNA is shown as sticks and balls.

• 

  Fig. 2. Interactions mediating RNA encapsidation. A The detailed interactions between N protein and RNA. The left panel shows an overall view of two protomers interacting with ssRNA. One protomer is represented as a cartoon, and the other as surface representation. The middle panel is a zoom-in view of the RNA binding cleft, highlighting essential residues involved in RNA binding. The right panel is a schematic representation of the RNA chain and the interacting residues. B The representation of surface electrostatic potential. The panel shows the protomers bound to ssRNA with the zoom-in views of the RNA binding cleft region. Residues Y258 and K178 involved in RNA chain flipping are indicated, along with a side view of the RNA-binding channel, showing size details.

• 

  Fig. 3. Protomer-protomer interactions. The interactions between two protomers are displayed. Left: overall view of two RNA-bound protomers, Chain K (blue) and Chain L (olive). A key hydrophobic core, which stabilizes the oligomeric state, is indicated by a transparent magenta oval, formed between the NT-arm helix 1 of one protomer (L) and the CT-Ncore helices 12 and 13 of another protomer (K). Right: zoom-in views of the interaction regions, highlighting important residues involved in hydrogen bonds and salt bridges, which are labeled and depicted as sticks.

• 

  Fig. 4. Structural comparisons within the Mononegavirales order. Superposition of NiV N structure with 10 N protein structures from three families within the Mononegavirales order: Paramyxoviridae family - SeV (PDB: 6M7D), MeV (PDB: 4UFT), HPIV3 (PDB: 7EV8), HPIV5 (PDB: 4XJN), MuV (PDB: 7EWQ), NDV (PDB: 6JC3), HEV (PDB: 8C4H); Filoviridae family - Ebola (PDB: 6C54); Pneumoviridae family - RSV (PDB: 8IUO), and HMPV (PDB: 8PDS). A Structural differences in the positioning of helix 5 of NT-Ncore. B Differences within the CT-Ncore and CT-arm regions. C RNA binding cleft, with important conserved residue positions indicated based on the NiV protomer model from this study. Key residues, including K178 and Y258, are crucial for RNA chain flipping. Additionally, Q199, R192, and R193 are conserved within the family and topologically conserved across the order (indicated by a gray circle). An exception is observed in the Pneumoviridae family, where helix 8 is bent, and the corresponding R198 is distant from the RNA.

• 

  Fig. 5. Functional validation of N-N, N-RNA and two-turn interfaces. A Overall view of two RNA-bound protomers with key residues mediating N–N (black) and N–RNA (red) interactions. B Low-pass filtered map (at 6 Å) showing density for potential inter-layer contacts. Loops involved in two-turn interactions are highlighted in red. C–E NanoLuc minigenome assays of NiV N mutants versus wild-type (WT). Data are shown as mean ± SD (n = 4). NC: negative control. Statistical significance was assessed by two-tailed unpaired t-test (*P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; ns, not significant). Source data are provided as Supplementary Data 1. F Focused multiple sequence alignment of Orthoparamyxoviridae N proteins, centered on the inter-turn loops. Sequences were obtained from UniProtKB: NiV, Nipah virus (Q9IK92); HeV, Hendra virus (O89339); CedV, Cedar virus (J7H328); GhV, Ghana virus (I0E088); LayV, Langya virus (A0AAX3C958); MojV, Mojiang virus (W8SIW1); MeV, Measles virus (P26029); HPIV3, Human parainfluenza virus type 3 (P06159); and SeV, Sendai virus (O57286).