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In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

Abstract

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid1,2,3, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome4,5,6,7; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus)8; it was the first fully sequenced organism9 and is a model system for studies of translational gene regulation10,11, RNA–protein interactions12,13,14, and RNA virus assembly15,16,17. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice18,19. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

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Figure 1: CryoEM asymmetric reconstruction of MS2 at 3.6 Å resolution.
Figure 2: Modelling the ssRNA genome.
Figure 3: Conserved interaction motifs between RNA stem–loops and coat protein dimers.
Figure 4: Maturation protein and its interactions with the 3′-end stem–loop.
Figure 5: Binding of maturation protein to genome and bacterial F-pilus.

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Acknowledgements

This project was supported in part by grants from the National Institutes of Health (GM071940, DE025567, DE023591, CA177322 and AI094386) and National Science Foundation (DMR-1548924). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines (supported by UCLA and by instrumentation grants from the NIH (1S10OD018111, 1U24GM116792) and NSF (DBI-1338135)). X.D. and Z.L. were supported in part by fellowships from the China Scholarship Council. We appreciate critical reading of the manuscript by F. Guo.

Author information

Authors and Affiliations

Authors

Contributions

X.D., Z.H.Z. and R.S. designed the project; X.D., Z.L. and S.S. prepared the sample and acquired cryoEM data; X.D. solved the structure; X.D. and M.L. built the model; X.D., M.L., Y.D., Z.H.Z. and R.S. interpreted the results; X.D. and Z.H.Z. wrote the paper; and all authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to Z. Hong Zhou or Ren Sun.

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The authors declare no competing financial interests.

Additional information

Reviewer Information

Nature thanks W. Dai and J. E. Johnson for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Resolution assessment of the cryoEM reconstruction.

a, ‘Gold-standard’ FSC curve of the cryoEM reconstruction. The average resolution of the final density map is 3.6 Å as determined by the FSC = 0.143 criterion40. b, Local resolution assessed by ResMap41. Density voxels are coloured according to their local resolution as defined in the colour scale on the right. Only half of the capsid is shown to expose the RNA densities inside. c, d, CryoEM densities of a coat protein dimer (c) or the maturation protein (d) with their bound RNA stem–loops to show quality of the density map. In both cases, the cryoEM densities are semitransparent to show the fitted atomic models of the protein and RNA.

Extended Data Figure 2 Three-dimensional classification.

The entire dataset of the cryoEM images were subjected to 3D classification and refinement starting from a single initial model of the asymmetric reconstruction. Ten classes were arbitrarily set. The resulting density maps were compared with the reconstruction of the whole dataset and with each other. The overall structures of the ten classes are almost identical, except for small regions as exemplified by the region enclosed in the dashed circle in the superimposed map. The RNA fragments of these regions have multiple conformations, and are thus not traced in our model. Overall, we were able to trace an RNA density amounting to 80% of the genome.

Extended Data Figure 3 Backbone model of MS2 genome segment 1–615.

Part of the traced backbone model of MS2 genome (top panel; rainbow-coloured blue to red from 5′ to 3′) is compared with the predicted secondary structure (bottom panel) of genome sequence 1615. Matching stem–loops in the two are marked with the same letter. Atomic models of high-resolution stem–loops (ribbons in top panel) contained in the segment are also shown. Some of the base pairings in the predicted secondary structure have been modified to make it more consistent with the observed structure. Dashed box in the bottom panel denotes flexible stem–loop that is not well resolved in the cryoEM density map and thus not traceable for the backbone.

Extended Data Figure 4 Backbone model of MS2 genome segment 881–1290.

Part of the traced backbone model of MS2 genome (top panel; rainbow-coloured blue to red from 5′ to 3′) is compared with the predicted secondary structure (bottom panel) of genome sequence 881–1290. Matching stem–loops in the two are marked with the same letter. Atomic models of high-resolution stem–loops (ribbons in top panel) contained in the segment are also shown. Some of the base pairings in the predicted secondary structure have been modified to make it more consistent with the observed structure.

Extended Data Figure 5 Backbone model of MS2 genome segment 1711–2340.

Part of the traced backbone model of MS2 genome (top panels; rainbow-coloured blue to red from 5′ to 3′) is compared with the predicted secondary structure (bottom panel) of genome sequence 1711–2340. Matching stem–loops in the two are marked with the same letter. Atomic models of high-resolution stem–loops (ribbons in top panels) contained in the segment are also shown. Some of the base pairings in the predicted secondary structure have been modified to make it more consistent with the observed structure.

Extended Data Figure 6 Backbone model of MS2 genome segment 2753–3388.

Part of the traced backbone model of MS2 genome (top panel; rainbow-coloured blue to red from 5′ to 3′) is compared with the predicted secondary structure (bottom panel) of genome sequence 2753–3388. Matching stem–loops in the two are marked with the same letter. Atomic models of high-resolution stem–loops (ribbons in top panel) contained in the segment are also shown. Some of the base pairings in the predicted secondary structure have been modified to make it more consistent with the observed structure. Dashed boxes in the bottom panel denote flexible stem–loops that are not well resolved in the cryoEM density map and thus not traceable for the backbone. Black wire in the top panel denotes RNA segment 2341–2359 that has long-range base-pairing interactions (also illustrated in Fig. 2d) with this segment, and the pairing bases are marked with black arc in the bottom panel.

Extended Data Figure 7 Backbone model of MS2 genome segment 3418–3569.

Part of the traced backbone model of MS2 genome (top panel; rainbow-coloured blue to red from 5′ to 3′) is compared with the predicted secondary structure (bottom panel) of genome sequence 3418–3569. Matching stem–loops in the two are marked with the same letter. Some of the base pairings in the predicted secondary structure have been modified to make it more consistent with the observed structure.

Extended Data Figure 8 Secondary structure of the MS2 genome.

Secondary structures of all genome segments in Fig. 2d and Extended Data Figs 3, 4, 5, 6, 7 are assembled to show the secondary structure of the entire MS2 genome. The genome sequences are coloured according to the genes encoded as depicted in the schematic diagram at the bottom, except for the lysis gene which overlaps with the coat protein gene and the replicase gene. The star signs denote the positions of the 16 high-resolution stem–loops. Segments enclosed with dotted boxes or ellipses are flexible.

Extended Data Figure 9 CryoEM densities (mesh) and atomic models (stick) of the 15 high-resolution RNA stem–loops that interact with coat protein dimers (ribbon).

Supplementary information

Supplementary Data 1 and 2

This zipped file contains 2 files showing (1) Secondary structure of the MS2 genome in FASTA file format and (2) Secondary structure of the MS2 genome in JSON file format. (ZIP 2903 kb)

Supplementary Data 3

This file shows the traced backbone of the MS2 genome. This backbone model was generated by manually tracing RNA densities in the MS2 asymmetric reconstruction low-pass filtered to 6Å resolution, using the “C-alpha Baton Mode” tool in Coot. It should be noticed that “C-alphas” in this model do not represent positions of individual ribonucleotides. Here, only the connected path of the C-alphas is meaningful and represents the path of the ssRNA chain. (TXT 311 kb)

CryoEM asymmetric reconstruction of MS2 virion

The video begins with the surface view of MS2 at 3.6Å resolution. The density map was then low-pass filtered to 6Å resolution, and half or all of the capsid shell was removed to expose the well-organized ssRNA genome packaged inside the capsid. (MP4 26188 kb)

3D classification reveals marginal flexibility of the MS2

3D classification of the cryoEM dataset produced 10 (arbitrarily set) classes. RNA densities in the asymmetric reconstruction from the entire dataset (the first one, radially coloured), or from each class, are shown one by one. They are also superimposed for comparison. Overall structure of the ssRNA genome is consistent among all classes, but some segments do not completely fit to each other in the 11 structures, indicating some level of flexibility. (MP4 13433 kb)

Interactions between a RNA stem-loop and a CP-dimer

Stem-loop 1746-1764 encompassing the start codon of the MS2 replicase gene is shown with the bound CP-dimer as an example to demonstrate the three conserved interaction motifs between RNA and capsid shell. (MP4 24655 kb)

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Dai, X., Li, Z., Lai, M. et al. In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus. Nature 541, 112–116 (2017). https://doi.org/10.1038/nature20589

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