Letter | Published:

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

Nature volume 541, pages 112116 (05 January 2017) | Download Citation

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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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

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

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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 interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Z. Hong Zhou or Ren Sun.

Reviewer Information

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

Extended data

Supplementary information

Zip files

  1. 1.

    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.

Text files

  1. 1.

    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.

Videos

  1. 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. 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. 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.

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DOI

https://doi.org/10.1038/nature20589

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