Figure 1.
Heterotrimeric VSV NΔ210-P dimer complex in solution.
The complex formed between NΔ21 and full-length P dimer in solution was analyzed by SEC-MALLS. The NΔ210-P complex elutes at 12.0 mL and the remaining NΔ210-P60 complex elutes at 15.6 mL (line). The molecular mass of 104±4 kDa (crosses) indicates a 1∶2 complex between one RNA-free NΔ21 and two P molecules in accordance with the previous observation that P forms exclusively dimers in solution and with the N0-P2 complex determined for rabies virus.
Figure 2.
Heterodimeric VSV NΔ210-P60 complex in solution.
(A) The absence of RNA in the complex is revealed by the UV absorbance spectrum, which exhibits an A280 nm/A260 nm ratio of 2.0. (B) Analysis of the NΔ210-P60 complex by SEC-MALLS. The complex elutes as a single peak at 15.6 mL (line), and the presence of the two proteins in the complex is demonstrated by SDS-PAGE analysis of the peak fraction using Coomassie blue staining (inset). The molecular mass of 53±2 kDa (crosses) indicates a 1∶1 complex between NΔ21 and P60 (calculated molecular mass = 45,377 Da (NΔ21)+8,053 Da (P60) = 53,430 Da). (C) Experimental SAXS data (open circles) up to 3.5 nm−1. The SAXS curve recorded at ESRF beamline ID 14-3 shows the scattering intensity I(q) as a function of the scattering vector, . (D) Average ab initio bead model of the NΔ210-P60 complex. The N protomer extracted from the circular N-RNA complex (2GIC chain E) fits to the SAXS-derived model, except for the NCT-loop.
Figure 3.
Crystal structure of a decameric form of the NΔ210-P60 complex.
(A) Overall structure of the decamer of NΔ210-P60 complex. NΔ21 is shown in green and P60 in red. (B) Ribbon representation of one protomer of NΔ210-P60. The central hinge region of N (aa 200–300) is shown in yellow and P60 (aa 6–35) is shown in red.
Table 1.
Data collection and refinement statistics (molecular replacement).
Figure 4.
P binding hinders RNA binding and self-assembly of soluble N.
(A) Representations of one protomer from the NΔ210-P60 complex. The N protomer is shown as space filling model in green and P60 is shown as a cartoon representation in red. The visible N- and C-terminal residues of P60 are labeled. (B) Representations of one protomer from the w.t. N-RNA complex. The 3′ terminal nucleotide of the RNA molecule is shown in yellow. These representations show that P60 fills the RNA-binding cavity on the side of N that accommodates the 3′-end of the RNA molecule. (C, D) Close-up of the interactions between exchangeable sub-domains in the circular NΔ210-P60 and N-RNA complexes. In the w.t. N-RNA complex (D), the NNT-arm of protomer Ni−1 contacts the NCT-loop of protomer Ni+1 while both sub-domains are docked on the back-side of protomer Ni (in green). In the NΔ210-P60 complex (C), the N-terminal extremity of P60 docks on protomer Ni (in green) at the position of the NNT-arm of protomer Ni−1 and contacts the NCT-loop of protomer Ni+1. These representations suggest that P60 interferes with the assembly of N in the absence of RNA.
Figure 5.
Surface properties and amino acid conservation in the P binding site.
(A) Close up of the interface between RNA-free NΔ21 and P60 showing the hydrophobic contacts and salt bridges. Residues 17 to 31 of P60 fold into an amphipathic α-helix that lies in a hydrophobic cavity formed by residues of the hinge region of N and is stabilized by hydrophobic contacts involving residues of P60 spaced i+3 or i+4 (Leu17, Val21, Ile24 and Ile27). Tyr14 docks into a small cavity lined with hydrophobic residues. Hydrophobic side chains in N are colored in yellow and hydrophobic side chains of P60 are labeled. The complex is also stabilized by salt bridges between Asp25 of P60 and Arg312 and His233 of N (in blue) and between Arg16 of P60 and Asp269 of N (in red). (B) Amino acid sequence conservation between VSV and RAV N. Identical residues are shown in dark blue and similar residues are shown in light blue. The surface area circled in black shows the binding groove of P and the surface area circle in red shows the hydrophobic site common to both P and the bases at the 3′ end of the RNA. (C, D) Electrostatic surface potential of the NΔ210 protein (C) compared with that of the NΔ210-P60 complex (D). Both panels show in the same orientations the two sides of the NΔ210 protein involved in binding the MoRE of P. The arrows indicate regions in which the electrostatic surface potential of N is modified by the presence of the peptide. The surface potentials were calculated with the Delphi program and are color-coded on the surface from red (negatively charged residues, −7 kcal/mol) to blue (positively charged residues, +7 kcal/mol).
Figure 6.
The N-terminal and C-terminal region of P60 flanking the MoRE exhibits conformational flexibility in the soluble complex.
(A) Comparison of the 2D 1H-15N HSQC NMR spectra of free 15N, 13C, 2H-labelled P60 (blue) and in complex with NΔ21 (red). Both spectra were recorded at 14.1 T and 25°C in 20 mM Tris-HCl, 150 mM NaCl, 50 mM Glu, 50 mM Arg with 10% D2O adjusted to pH 6.0. The labels indicate the assignment of the resonances of the complex. (B) 15N R2 spin relaxation rates measured under the same conditions as (A) for the free 15N-labeled P60 (blue) and the NΔ210-P60 complex (red). (C) 15N R2 spin relaxation rates measured at 14.1 T and 10°C of the free 15N-labeled P60 (blue), a mixture of 0.27 mM 15N-labeled P60 and 0.09 mM unlabeled MBP-NΔ21 (green) and a mixture of 0.24 mM 15N-labeled P60 and 0.17 mM unlabeled MBP-NΔ21 (red).
Figure 7.
Schematic representations of the mechanism of RNA replication of VSV.
The nucleoprotein (in green) forms with the RNA genome (blue line) the active template for the polymerase complex comprising the L (in yellow) and P (in red) proteins. (A) Encapsidation during RNA replication. During replication, the newly synthesized antigenomic or genomic RNA is encapsidated by nascent N molecules that are transferred from the soluble N0-P complex. In the N0-P complex, the N-terminal MoRE of P prevents host-cell RNA binding by obstructing the RNA binding groove and the self-assembly of N by interfering with the docking of the NNT-arm of another N. Upon the transfer of N to the growing viral RNA P is released, the binding groove for the NNT-arm is freed in the RNA-bound form and can accept the next incoming N molecule. (B) Initiation of RNA synthesis. By binding at the 3′ extremity of the nucleocapsid, the N-terminal MoRE of P might displace nucleotides from the N molecule and allow the polymerase to initiate RNA synthesis.