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

Journal name:
Nature
Volume:
541,
Pages:
112–116
Date published:
DOI:
doi:10.1038/nature20589
Received
Accepted
Published online

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.

At a glance

Figures

  1. CryoEM asymmetric reconstruction of MS2 at 3.6 Å resolution.
    Figure 1: CryoEM asymmetric reconstruction of MS2 at 3.6 Å resolution.

    a, b, Front (a) and back (b) views of the cryoEM density map along an icosahedral two-fold symmetry axis with some two-, three- and five-fold axes indicated. The capsid shell is radially coloured with the maturation protein highlighted in magenta and the ssRNA genome inside the capsid in blue. c, Cut-open view with half of the capsid shell removed to expose the genome. d, Segmented cryoEM densities (mesh) superimposed with their corresponding atomic models (sticks). Top and bottom left, typical β-strand and α-helix densities, respectively, from the maturation protein. Bottom right, part of a maturation protein-bound RNA stem–loop. Purines and pyrimidines are readily distinguishable.

  2. Modelling the ssRNA genome.
    Figure 2: Modelling the ssRNA genome.

    a, Backbone structure of the genome (wire) and non-uniform distribution of the high-resolution stem–loops (ribbons). Backbone is rainbow-coloured (blue to red) from 5′ to 3′. bd, Example of tracing RNA backbone. Part of the genome density (grey in b) is segmented out and superimposed with its backbone model (rainbow-coloured wire, blue to red from 5′ to 3′; b, c). For each of the two high-resolution stem–loops (ribbons in c) contained in this segment, a degenerate sequence was derived on the basis of the resolved bases and used to search against the genome to identify sequence candidates. Each of these short sequence candidates was expanded in both directions to include about 500 bases for secondary structure prediction. The predicted secondary structure was then correlated with the backbone obtained in b and only one of these sequence candidates yielded the correct sequence registration of individual stem–loops (indicated by letters Q–W in c, d). The backbone model reveals kissing-loop and long-range base-pairing interactions as indicated.

  3. Conserved interaction motifs between RNA stem–loops and coat protein dimers.
    Figure 3: Conserved interaction motifs between RNA stem–loops and coat protein dimers.

    a, Secondary structures of RNA stem–loops with nucleotides involved in the three types of conserved interaction motif coloured. Letters beneath some of the stem–loops identify panels in which the atomic model for that stem–loop is shown. be, Atomic model of stem–loop 1747–1763 and its interactions with a coat protein dimer (pink and sky blue ribbons). In c, positively charged or polar residues of the coat protein dimer interacting with phosphates of the RNA backbone (sticks) are indicated. Expanded views of the stem (d) and loop (e) regions show the interaction motifs conserved among the 15 stem–loops. fj, Accommodation of diversities in sequence or local environment. Stem–loop 2781–2796 (f), viewed in the same orientation as in d, shows that a guanine forms the same kind of hydrogen bond with Thr45 and Ser47 as an adenine in d. Stem–loops 977–990 (g) and 102–114 (h), viewed in the same orientation as in e, show that a purine (G983 in g) instead of a pyrimidine (U1756 in e) stacks with Tyr85 and that a pyrimidine (C109 in h) forms only one hydrogen bond instead of a purine (A1757 in e) forming two hydrogen bonds with Thr45 and Ser47. Stem–loop 179–200 (i, j) binds to a coat protein dimer from a very different angle owing to steric hindrance of a neighbouring stem–loop (not shown). Nonetheless, the RNA fold and one of the three interaction motifs are conserved, although a hydrogen bond is formed with Thr59 instead of Ser47.

  4. Maturation protein and its interactions with the 3′-end stem–loop.
    Figure 4: Maturation protein and its interactions with the 3′-end stem–loop.

    a, Incorporation of maturation protein into the capsid shell. The maturation protein (magenta) replaces a coat protein dimer at a two-fold symmetry axis and induces structural changes of neighbouring coat proteins. Atomic models of the changed coat proteins (coloured ribbons) are superimposed with those of coat proteins at other two-fold symmetry axes (beige ribbons) that are unaffected by the maturation protein. b, Maturation protein model, rainbow-coloured (blue to red from N to C terminus). c, Binding of the 3′-end stem–loop to the maturation protein and neighbouring coat proteins as viewed inside the capsid. d, Base-pairing in the 3′-end stem–loop as observed in our structure interacting with maturation protein (right), or theoretically as a free stem–loop (left). eh, Details of the interactions between the 3′-end stem–loop and the maturation protein or coat proteins. h, Expanded view of the RNA stem region with half of the stem hidden for clarity.

  5. Binding of maturation protein to genome and bacterial F-pilus.
    Figure 5: Binding of maturation protein to genome and bacterial F-pilus.

    a, Overview of maturation protein (magenta) with surrounding RNA stem–loops (wires coloured as in Fig. 2a) shown in the same orientation as in Fig. 3c. b, As in a but without the RNA backbone model, showing the distribution of positively charged arginine and lysine residues in the maturation protein that bind RNA stem–loops. c, d, Fitting of our atomic models (ribbons) of the MS2 virion into a tomographic reconstruction (EMD-2365, semitransparent surface) of MS2 attached to bacterial F-pilus19. The maturation protein projects obliquely out from the capsid surface, resulting in a slight tilt (approximately 9°) of the MS2 virion when attached to the F-pilus (d).

  6. Resolution assessment of the cryoEM reconstruction.
    Extended Data Fig. 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.

  7. Three-dimensional classification.
    Extended Data Fig. 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.

  8. Backbone model of MS2 genome segment 1–615.
    Extended Data Fig. 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.

  9. Backbone model of MS2 genome segment 881–1290.
    Extended Data Fig. 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.

  10. Backbone model of MS2 genome segment 1711–2340.
    Extended Data Fig. 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.

  11. Backbone model of MS2 genome segment 2753–3388.
    Extended Data Fig. 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.

  12. Backbone model of MS2 genome segment 3418–3569.
    Extended Data Fig. 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.

  13. Secondary structure of the MS2 genome.
    Extended Data Fig. 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.

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

Videos

  1. CryoEM asymmetric reconstruction of MS2 virion
    Video 1: 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.
  2. 3D classification reveals marginal flexibility of the MS2
    Video 2: 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.
  3. Interactions between a RNA stem-loop and a CP-dimer
    Video 3: 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.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

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Author information

Affiliations

  1. Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA), Los Angeles, California 90095, USA

    • Xinghong Dai,
    • Sara Shu,
    • Yushen Du &
    • Ren Sun
  2. The California NanoSystems Institute (CNSI), UCLA, Los Angeles, California 90095, USA

    • Xinghong Dai,
    • Zhihai Li,
    • Z. Hong Zhou &
    • Ren Sun
  3. Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California 90095, USA

    • Zhihai Li,
    • Mason Lai &
    • Z. Hong Zhou
  4. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China

    • Zhihai Li

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Reviewer Information

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

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Resolution assessment of the cryoEM reconstruction. (926 KB)

    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.

  2. Extended Data Figure 2: Three-dimensional classification. (740 KB)

    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.

  3. Extended Data Figure 3: Backbone model of MS2 genome segment 1–615. (426 KB)

    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.

  4. Extended Data Figure 4: Backbone model of MS2 genome segment 881–1290. (316 KB)

    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.

  5. Extended Data Figure 5: Backbone model of MS2 genome segment 1711–2340. (458 KB)

    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.

  6. Extended Data Figure 6: Backbone model of MS2 genome segment 2753–3388. (494 KB)

    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.

  7. Extended Data Figure 7: Backbone model of MS2 genome segment 3418–3569. (363 KB)

    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.

  8. Extended Data Figure 8: Secondary structure of the MS2 genome. (597 KB)

    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.

  9. 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). (1,291 KB)

Supplementary information

Video

  1. Video 1: CryoEM asymmetric reconstruction of MS2 virion (25.57 MB, Download)
    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.
  2. Video 2: 3D classification reveals marginal flexibility of the MS2 (13.11 MB, Download)
    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.
  3. Video 3: Interactions between a RNA stem-loop and a CP-dimer (24.07 MB, Download)
    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.

Zip files

  1. Supplementary Data 1 and 2 (2.8 MB)

    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.

Text files

  1. Supplementary Data 3 (311 KB)

    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.

Additional data