Letter | Published:

Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus


Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1,2,3,4,5,6,7,8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10,11,12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10–12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11,12,13,14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

Density maps and the model that support the findings of this study have been deposited in the Electron Microscopy Data Bank and in the Protein Data Bank (PDB) with the accession codes EMD-0299 and PDB 6HY0 (nucleocapsid, icosahedral symmetry); EMD-0300 (nucleocapsid, D3 symmetry); EMD-0301 (genome first-layer organization pD3); EMD-0302 (genome first-layer organization pD3′), EMD-0303 (genome first-layer organization pC2); EMD-0304 (genome first-layer sub-organization pD3′-1); EMD-0305 (genome first-layer sub-organization pD3′-2); and EMD-0306 (genome first-layer sub-organization pD3′-3). Maps from the layer-by-layer genome reconstruction have been submitted with the accession codes EMD-0294 (genome second layer), EMD-0295 (genome third layer) and EMD-0296 (genome fourth and fifth layers).

Code availability

Custom software code used in this study is available from the corresponding author upon request and from https://github.com/OPIC-Oxford.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Speir, J. A. & Johnson, J. E. Nucleic acid packaging in viruses. Curr. Opin. Struct. Biol. 22, 65–71 (2012).

  2. 2.

    Black, L. W. DNA packaging in dsDNA bacteriophages. Annu. Rev. Microbiol. 43, 267–292 (1989).

  3. 3.

    Booy, F. P. et al. Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus. Cell 64, 1007–1015 (1991).

  4. 4.

    Lepault, J., Dubochet, J., Baschong, W. & Kellenberger, E. Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples. EMBO J. 6, 1507–1512 (1987).

  5. 5.

    Smith, D. E. et al. The bacteriophage ɸ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001).

  6. 6.

    Cerritelli, M. E. et al. Encapsidated conformation of bacteriophage T7 DNA. Cell 91, 271–280 (1997).

  7. 7.

    Jiang, W. et al. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439, 612–616 (2006).

  8. 8.

    LaMarque, J. C., Le, T.-V. L. & Harvey, S. C. Packaging double-helical DNA into viral capsids. Biopolymers 73, 348–355 (2004).

  9. 9.

    Roers, A., Hiller, B. & Hornung, V. Recognition of endogenous nucleic acids by the innate immune system. Immunity 44, 739–754 (2016).

  10. 10.

    Patton, J. T. & Spencer, E. Genome replication and packaging of segmented double-stranded RNA viruses. Virology 277, 217–225 (2000).

  11. 11.

    Gouet, P. et al. The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography. Cell 97, 481–490 (1999).

  12. 12.

    Yazaki, K. & Miura, K. Relation of the structure of cytoplasmic polyhedrosis virus and the synthesis of its messenger RNA. Virology 105, 467–479 (1980).

  13. 13.

    Liu, H. & Cheng, L. Cryo-EM shows the polymerase structures and a nonspooled genome within a dsRNA virus. Science 349, 1347–1350 (2015).

  14. 14.

    Zhang, X. et al. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus. Nature 527, 531–534 (2015).

  15. 15.

    Day, L. A. & Mindich, L. The molecular weight of bacteriophage ɸ6 and its nucleocapsid. Virology 103, 376–385 (1980).

  16. 16.

    Sen, A. et al. Initial location of the RNA-dependent RNA polymerase in the bacteriophage ɸ6 procapsid determined by cryo-electron microscopy. J. Biol. Chem. 283, 12227–12231 (2008).

  17. 17.

    Sun, X., Bamford, D. H. & Poranen, M. M. Probing, by self-assembly, the number of potential binding sites for minor protein subunits in the procapsid of double-stranded RNA bacteriophage ɸ6. J. Virol. 86, 12208–12216 (2012).

  18. 18.

    Sun, Z. et al. Double-stranded RNA virus outer shell assembly by bona fide domain-swapping. Nat. Commun. 8, 14814 (2017).

  19. 19.

    Nemecek, D., Qiao, J., Mindich, L., Steven, A. C. & Heymann, J. B. Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage ɸ6 procapsid. J. Virol. 86, 11616–11624 (2012).

  20. 20.

    Ilca, S. L. et al. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes. Nat. Commun. 6, 8843 (2015).

  21. 21.

    Ikonen, T., Kainov, D., Timmins, P., Serimaa, R. & Tuma, R. Locating the minor components of double-stranded RNA bacteriophage ɸ6 by neutron scattering. J. Appl. Cryst. 36, 525–529 (2003).

  22. 22.

    Huiskonen, J. T. et al. Structure of the bacteriophage ɸ6 nucleocapsid suggests a mechanism for sequential RNA packaging. Structure 14, 1039–1048 (2006).

  23. 23.

    Earnshaw, W. C. & Harrison, S. C. DNA arrangement in isometric phage heads. Nature 268, 598–602 (1977).

  24. 24.

    Strzelecka, T. E., Davidson, M. W. & Rill, R. L. Multiple liquid crystal phases of DNA at high concentrations. Nature 331, 457–460 (1988).

  25. 25.

    Livolant, F. & Leforestier, A. Condensed phases of DNA: structures and phase transitions. Prog. Polym. Sci. 21, 1115–1164 (1996).

  26. 26.

    Rey, A. D. Liquid crystal models of biological materials and processes. Soft Matter 6, 3402–3429 (2010).

  27. 27.

    Morávek, Z., Neidle, S. & Schneider, B. Protein and drug interactions in the minor groove of DNA. Nucleic Acids Res. 30, 1182–1191 (2002).

  28. 28.

    Wang, X. et al. Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly. Proc. Natl Acad. Sci. USA 115, 7344–7349 (2018).

  29. 29.

    Van Etten, J. L., Burbank, D. E., Cuppels, D. A., Lane, L. C. & Vidaver, A. K. Semiconservative synthesis of single-stranded RNA by bacteriophage ɸ6 RNA polymerase. J. Virol. 33, 769–773 (1980).

  30. 30.

    Skehel, J. J. & Joklik, W. K. Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores. Virology 39, 822–831 (1969).

  31. 31.

    Usala, S. J., Brownstein, B. H. & Haselkorn, R. Displacement of parental RNA strands during in vitro transcription by bacteriophage ɸ6 nucleocapsids. Cell 19, 855–862 (1980).

  32. 32.

    Makeyev, E. V. & Bamford, D. H. The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 19, 6275–6284 (2000).

  33. 33.

    Patton, J. T., Jones, M. T., Kalbach, A. N., He, Y. W. & Xiaobo, J. Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. J. Virol. 71, 9618–9626 (1997).

  34. 34.

    Tortorici, M. A., Broering, T. J., Nibert, M. L. & Patton, J. T. Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus. J. Biol. Chem. 278, 32673–32682 (2003).

  35. 35.

    Petrov, A. S. & Harvey, S. C. Packaging double-helical DNA into viral capsids: structures, forces, and energetics. Biophys. J. 95, 497–502 (2008).

  36. 36.

    Vidaver, A. K., Koski, R. K. & Van Etten, J. L. Bacteriophage ɸ6: a lipid-containing virus of Pseudomonas phaseolicola. J. Virol. 11, 799–805 (1973).

  37. 37.

    Bamford, D. H., Ojala, P. M., Frilander, M., Walin, L. & Bamford, J. K. H. in Microbial Gene Techniques (Methods in Molecular G enetics Vol. 6) (ed. Adolph, K. W.) 455–474 (Academic, San Diego, 1995).

  38. 38.

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  39. 39.

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

  40. 40.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  41. 41.

    Bai, X.-C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

  42. 42.

    Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).

  43. 43.

    Nicholls, R. A., Long, F. & Murshudov, G. N. Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012).

  44. 44.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

  45. 45.

    DiMaio, F. et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015).

  46. 46.

    da Fontoura Costa, L., Rocha, F. & Araújo de Lima, S. M. Characterizing polygonality in biological structures. Phys. Rev. E 73, 011913 (2006).

  47. 47.

    Goodsell, D. S. & Dickerson, R. E. Bending and curvature calculations in B-DNA. Nucleic Acids Res. 22, 5497–5503 (1994).

Download references


We thank R. Tarkiainen for technical assistance and Diamond Light Source for access to and support with the cryo-EM facilities (EM14856) at the UK national electron bio-imaging centre (eBIC), which is funded by the Wellcome Trust, MRC and BBSRC. We acknowledge the use of the Instruct-HiLIFE Biocomplex unit (University of Helsinki and Instruct-FI), and the support of the Academy of Finland (grant 1306833) for the unit. This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (649053 to J.T.H.); a Wellcome Trust administrative support grant (203141/Z/16/Z); a Wellcome Trust four-year PhD studentship (109135/Z/15/A to S.L.I.); the Academy of Finland (grant 272507 to M.M.P.); and the Sigrid Jusélius Foundation (to M.M.P).

Reviewer information

Nature thanks Alex Evilevitch and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

S.L.I. reconstructed the genome maps and built the genome models. X.S. prepared the virus sample. A.K. reconstructed the nucleocapsid map. F.d.H. assisted with data collection. F.D. provided custom tools and, together with J.M.G., gave advice for genome building. J.M.G., D.I.S., M.M.P. and J.T.H. provided supervision. K.E.O. analysed RNA–protein contacts. J.T.H. and S.L.I. analysed the data, prepared the figures and wrote the manuscript. All authors commented on the manuscript.

Competing interests

F.d.H. and A.K. are both employees of Thermo Fisher Scientific.

Correspondence to Juha T. Huiskonen.

Extended data figures and tables

  1. Extended Data Fig. 1 Asymmetric reconstruction of the ɸ6 genome.

    a, The icosahedral symmetry (I1) in the ɸ6 nucleocapsid reconstruction, calculated from the original cryo-EM single particles (particle images), was relaxed to allow asymmetric reconstruction. The protein shells were subtracted, and the resulting genome-only particle images were used to calculate a D3 genome reconstruction in addition to asymmetric reconstructions that had either pD3 or pC2 symmetry. Three sub-conformations of the pD3′ reconstruction (pD3′-1, pD3′-2 and pD3′-3) are shown, rotated as indicated to reveal their differences in RNA organization. The names of the reconstructions that were used to model the layers of the dsRNA genome and dsRNA–P1 interactions are in bold and are indicated with dotted lines. bf, The Fourier shell correlation (FSC) is plotted for the reconstructions that are included in further analysis, namely I1, D3, pD3, pC2 and pD3′-1. Volume masking (blue line) was used to focus the FSC test on the area used in further modelling and this increased the estimated resolution compared to the original unmasked reconstruction (grey line). Possible effects of the masking were compensated for by noise randomization (red line), to create the final FSC curve (black line). The resolution at which the correlation drops below the FSC = 0.143 threshold is indicated. Source data

  2. Extended Data Fig. 2 Model of the ɸ6 genome.

    The cryo-EM asymmetric reconstruction (Density) and the model built into the same density (Model) are shown for each of the genome layers. The layers are coloured as in Fig. 1. The density shown for layer 1 is from the pD3′-1 map and the density for layers 2–5 is from the pD3 map.

  3. Extended Data Fig. 3 Modes of dsRNA packing in ɸ6.

    A histogram with the relative fractions of base pairs that are located in hexagonal, cubic and undefined packing regions. The total number of base pairs defined for each layer is provided. Source data

  4. Extended Data Fig. 4 Distances between different types of P1 residues and RNA.

    a, b, The fractions of different P1 amino acid residue types (hydrophobic, polar, positively charged and negatively charged) at different distances from the RNA are shown for the pD3 (a) and pC2 (b) models. Source data

  5. Extended Data Fig. 5 P1–RNA distances in the first genome layer with pD3 symmetry.

    The frequency of P1 residues in the pD3 conformation that have non-hydrogen atoms located at a shorter distance than 14 Å from non-hydrogen atoms in the dsRNA model is given in each distance bin (1–14 Å) for the P1 chains A and B together and separately. The total number of residues with an atom or atoms closer than the cut-off distance is given for each table. The frequencies are colour coded so that higher frequencies are in a lighter shade of orange and lower frequencies are darker. The difference in the total numbers between chains A and B is given (Comparison) as a log2 ratio (that is, the base-2 logarithm of the ratio of total frequencies for chains A and B). For example, a value of 1 means that chain A has 2 times as many included residues as chain B, and a value of 2 means that chain A has 4 times as many included residues as chain B (negative values mean that chain B had more included residues). Positive values are in red and negative values in blue. Those cases in which an included residue was present only in one of the two chains are indicated. Source data

  6. Extended Data Fig. 6 P1–RNA distances in the first genome layer with pC2 symmetry.

    The frequency of P1 residues in the pC2 conformation that have non-hydrogen atoms located at a shorter distance than 14 Å from non-hydrogen atoms in the dsRNA model is given in each distance bin (1–14 Å) for P1 chains A and B together and separately. The annotations are as in Extended Data Fig. 5. Source data

  7. Extended Data Table 1 Cryo-EM data collection and nucleocapsid model
  8. Extended Data Table 2 Cryo-EM reconstruction parameters

Supplementary information

  1. Reporting Summary

Source data

  1. Source Data Fig. 1

  2. Source Data Fig. 2

  3. Source Data Extended Data Fig. 1

  4. Source Data Extended Data Fig. 3

  5. Source Data Extended Data Fig. 4

  6. Source Data Extended Data Fig. 5

  7. Source Data Extended Data Fig. 6

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Structure of the ɸ6 nucleocapsid and dsRNA genome.
Fig. 2: Organization of the dsRNA genome.
Fig. 3: Organizational heterogeneity in the genome.
Fig. 4: Putative interactions between RNA and the P1 shell.
Extended Data Fig. 1: Asymmetric reconstruction of the ɸ6 genome.
Extended Data Fig. 2: Model of the ɸ6 genome.
Extended Data Fig. 3: Modes of dsRNA packing in ɸ6.
Extended Data Fig. 4: Distances between different types of P1 residues and RNA.
Extended Data Fig. 5: P1–RNA distances in the first genome layer with pD3 symmetry.
Extended Data Fig. 6: P1–RNA distances in the first genome layer with pC2 symmetry.


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.