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. 2019 Mar 5;116(10):4256-4264.
doi: 10.1073/pnas.1816417116. Epub 2019 Feb 20.

Assembly and cryo-EM Structures of RNA-specific Measles Virus Nucleocapsids Provide Mechanistic Insight Into Paramyxoviral Replication

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Free PMC article

Assembly and cryo-EM Structures of RNA-specific Measles Virus Nucleocapsids Provide Mechanistic Insight Into Paramyxoviral Replication

Ambroise Desfosses et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

Keywords: NMR; assembly; cryoelectron microscopy; measles virus; nucleocapsids.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cryo-EM structure of NCLP6A and NCLP5′. (A and B) Representative cryo-EM micrographs of NCLP6A (A) and NCLP5′ (B), with a representative class average shown as Inset. (Scale bar: 20 nm.) (C and D) Isosurface representation of the cryo-EM maps of NCLP6A (C) and NCLP5′ (D) colored as in the previously published work (11): NTDARM in blue navy, NTD in blue, CTD in salmon, CTDARM in yellow, RNA in green. A front view and a cutaway view are shown. (E and F) Zoom of three consecutive protomers of NCLP6A (E) and NCLP5′ (F) viewed from the helix exterior. Each RNA molecule is colored in different shades of green and the gaps between the RNA molecules are highlighted by red arrows. (G and H) Zoom of protein–RNA interaction in NCLP6A (G) and NCLP5′ (H), with asterisks to highlight gaps between the RNA molecules. Some clearly visible side chains described in the text are indicated.
Fig. 2.
Fig. 2.
Cryo-EM structure of RNA and NCLP. (A) Conformation of RNA in NCLP5′ and NCLP6A showing clear nucleobase stacking interactions between triplets comprising (6-1-2) and (3-4-5) that point toward solvent and protein, respectively. The gap between nucleotides 6 and 1 (asterisk) is evident in both structures. Weaker density is also seen at the C5′-O5′ atoms comprising the switch between the triplets (6-1-2) and (3-4-5) (orange arrows) but not between (3-4-5) and (6-1-2) (blue dashed arrows). (B) Representation of three N protomers within the NCLPs. Ni is represented in ribbon format (NCLP5′ and NCLP6A are superimposed and shown in orange and blue, respectively). The neighboring N molecules are shown in accessible surface format. The five phosphate groups of the RNA molecule bound to protomer Ni are shown as red spheres, while the position of the equivalent phosphate group at position one in the NCLPs assembled in vivo is shown in yellow (11) (PDB ID code 4UFT, absent in the NCLPs assembled in vitro). The orange arrow indicates the three accessible nucleotides in the final protomer of the assembled NCLP that are not buried in the cleft formed by a neighboring protomer. (C) Depiction of the N-terminal (blue) and C-terminal (pink) domains of Ni shown relative to the position of the bound RNA (orange), illustrating the overhang at the 3′ end. Residues 377–396 are not shown for clarity. The orange arrow again indicates the three accessible nucleotides.
Fig. 3.
Fig. 3.
Differences between NCLP5′ structures and N°P complexes. (A) N-terminal domains of N from PIV5, within the N0P complex (23), and within the crystallized ring structure of N:RNA (15), NiV N0P complex (20), and MeV N0P (21) (PDB ID codes 5WKN, 4XJN, 4CO6, and 5E4V, respectively) and NCLP5′ (present study). The α6-helix is shown in red (MeV numbering 186–203). (B) Density of the α6-helix. Orientation is ∼90°(x axis) compared with the same structures in A. (C) Comparison of the CTDs of MeV N0P (21) (green) and NCLP5′ (pink, present study). The induced helical turn between residues S346 and L350 is indicated. Further conformational rearrangements associated with domain reorientation upon NCLP formation are seen at the lower limb of the domain.
Fig. 4.
Fig. 4.
N–RNA interaction sites within the binding groove in NCLP6A. (AC) RNA-binding forms a cavity with amino acids from adjacent protomers. Adjacent protomers are shown in green and blue (ribbon) for the polyA NCLP. Bound RNA is shown in stick representation. The three bases pointing into the cavity are colored in red, while 1, 2, and 6 are colored in orange. (A) Cavities were calculated using a probe with radius 3 Å. Only one cavity (gray) is found. (B) Cavity surface is colored as a function of the amino acids in closest proximity (green and blue for adjacent protomers) (C) shows the view in B rotated by 90°. (D) Key interactions between a single polyA RNA molecule and a single nucleoprotein within the NCLP. RNA is shown in orange, and sites that were selected for mutation are shown in red (Q202, R195, E263, and N351). Sidechains of interacting protein residues are shown as sticks. Structures of protein and RNA in NCLP assembled on 5′ viral RNA are very similar to the conformation shown here (NCLP6A). The Inset shows the bipartite interaction of R195 with both backbone and sidechain of the nucleotide in position 5 (A in polyA).
Fig. 5.
Fig. 5.
Assembly of NCs from N0P. (A) Overlay of 1H-15N SOFAST HMQC experiments of P50N405 wild-type before (red) and 6 h after addition of HO-ACCAGA-OH (blue) showing the release of the P50 peptide giving rise to additional NMR signals. (B) Overlay of 1H-15N SOFAST HMQC experiments of N0P wild-type in the absence of RNA (red) and of N0P R195A 6 h after addition of HO-ACCAGA-OH (blue) demonstrating the absence of NCLP assembly. Note that the folded core domain of N is not visible in the spectrum. The mutated and the wild-type P1–50N1–405 spectra in the absence of RNA are therefore the same. (C and D) Zoom-ins into the respective spectra. The assignment of representative peaks corresponding to N and P residues are indicated in both zooms.
Fig. 6.
Fig. 6.
Kinetics of NC assembly. (A) Peak intensities from the 1H-15N SOFAST HMQC experiments during NCLP assembly were measured and plotted as a function of time. A set of peaks corresponding to P residues, which appear during assembly, were fitted with a monoexponential increase and then averaged. The average rate constants and corresponding SDs are plotted for each N0P mutant. The wild-type (wt) was repeated to assess reliability of the approach. Fits of representative kinetic traces (black: data points; red: exponential fit) of the wild-type and N351A assembly are shown in B and C, respectively.
Fig. 7.
Fig. 7.
The 3′ RNA is accessible to the RNA-dependent RNA polymerase complex. Representation of the possible positioning of the polymerase complex relative to the solvent accessible 3′ end of the RNA genome (orange surface) at the terminus of the NC (adjacent N protomers are shown in different shades of blue). Only NTAIL of the terminal N protomer is shown for simplicity. The protomer can bind P via two possible mechanisms, via an interaction between the NTAIL molecular recognition element and the XD domain of P, and via the N-terminal peptide of P that could bind to N, due to the absence of adjacent N protomers that displace all other P peptides in the NC. The unfolded domains PNTD and NTAIL were generated using the flexible-meccano algorithm (56). The conformation of the polymerase (L) is taken from the structure of the homologous enzyme from VSV (57). The binding of L to P is unknown and is shown figuratively here.

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