Visualizing virus assembly intermediates inside marine cyanobacteria (original) (raw)

Nature volume 502, pages 707–710 (2013)Cite this article

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Abstract

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET)1,2. This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

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Accessions

Electron Microscopy Data Bank

Data deposits

The averaged density maps of the procapsid, expanded capsid and the DNA-containing capsid have been deposited in the EBI under accession codes EMD-5742, EMD-5743, EMD-5744, EMD-5745 and EMD-5746, respectively.

References

  1. Danev, R. & Nagayama, K. Phase plates for transmission electron microscopy. Methods Enzymol. 481, 343–369 (2010)
    Article Google Scholar
  2. Murata, K. et al. Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure 18, 903–912 (2010)
    Article CAS Google Scholar
  3. Fuller, N. J. et al. Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the red sea. Appl. Environ. Microbiol. 69, 2430–2443 (2003)
    Article CAS Google Scholar
  4. Rocap, G., Distel, D. L., Waterbury, J. B. & Chisholm, S. W. Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S–23S ribosomal DNA internal transcribed spacer sequences. Appl. Environ. Microbiol. 68, 1180–1191 (2002)
    Article CAS Google Scholar
  5. Taylor, K. A. & Glaeser, R. M. Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. J. Struct. Biol. 163, 214–223 (2008)
    Article CAS Google Scholar
  6. Danev, R., Glaeser, R. M. & Nagayama, K. Practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy. Ultramicroscopy 109, 312–325 (2009)
    Article CAS Google Scholar
  7. Marko, M., Leith, A., Hsieh, C. & Danev, R. Retrofit implementation of Zernike phase plate imaging for cryo-TEM. J. Struct. Biol. 174, 400–412 (2011)
    Article CAS Google Scholar
  8. Rochat, R. H. et al. Seeing the portal in herpes simplex virus type 1 B capsids. J. Virol. 85, 1871–1874 (2011)
    Article CAS Google Scholar
  9. Liberton, M., Austin, J. R., II, Berg, R. H. & Pakrasi, H. B. Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography. Plant Physiol. 155, 1656–1666 (2011)
    Article CAS Google Scholar
  10. Ting, C. S., Hsieh, C., Sundararaman, S., Mannella, C. & Marko, M. Cryo-electron tomography reveals the comparative three-dimensional architecture of Prochlorococcus, a globally important marine cyanobacterium. J. Bacteriol. 189, 4485–4493 (2007)
    Article CAS Google Scholar
  11. Iancu, C. V. et al. Organization, structure, and assembly of α-carboxysomes determined by electron cryotomography of intact cells. J. Mol. Biol. 396, 105–117 (2010)
    Article CAS Google Scholar
  12. Schmid, M. F. et al. Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. J. Mol. Biol. 364, 526–535 (2006)
    Article CAS Google Scholar
  13. Pope, W. H. et al. Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a “horned” bacteriophage of marine synechococcus. J. Mol. Biol. 368, 966–981 (2007)
    Article CAS Google Scholar
  14. Chang, J. T. et al. Visualizing the structural changes of bacteriophage Epsilon15 and its Salmonella host during infection. J. Mol. Biol. 402, 731–740 (2010)
    Article CAS Google Scholar
  15. Hu, B., Margolin, W., Molineux, I. J. & Liu, J. The bacteriophage t7 virion undergoes extensive structural remodeling during infection. Science 339, 576–579 (2013)
    Article ADS CAS Google Scholar
  16. Schmid, M. F. Single-particle electron cryotomography (cryoET). Adv. Protein Chem. Struct. Biol. 82, 37–65 (2011)
    Article CAS Google Scholar
  17. Schmid, M. F. & Booth, C. R. Methods for aligning and for averaging 3D volumes with missing data. J. Struct. Biol. 161, 243–248 (2008)
    Article Google Scholar
  18. Raytcheva, D. A., Haase-Pettingell, C., Piret, J. M. & King, J. A. Intracellular assembly of cyanophage Syn5 proceeds through a scaffold-containing procapsid. J. Virol. 85, 2406–2415 (2011)
    Article CAS Google Scholar
  19. Chen, D. H. et al. Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus. Proc. Natl Acad. Sci. USA 108, 1355–1360 (2011)
    Article ADS CAS Google Scholar
  20. Prevelige, P. E., Thomas, D. & King, J. Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro . J. Mol. Biol. 202, 743–757 (1988)
    Article CAS Google Scholar
  21. Hegde, S., Padilla-Sanchez, V., Draper, B. & Rao, V. B. Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation. J. Virol. 86, 4046–4057 (2012)
    Article CAS Google Scholar
  22. Aksyuk, A. A. & Rossmann, M. G. Bacteriophage assembly. Viruses 3, 172–203 (2011)
    Article CAS Google Scholar
  23. Hendrix, R. W. Bacteriophage assembly. Nature 277, 172–173 (1979)
    Article ADS CAS Google Scholar
  24. Fuller, D. N. et al. Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations. J. Mol. Biol. 373, 1113–1122 (2007)
    Article CAS Google Scholar
  25. Machado, I. M. & Atsumi, S. Cyanobacterial biofuel production. J. Biotechnol. 162, 50–56 (2012)
    Article CAS Google Scholar
  26. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visulasualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996)
    Article CAS Google Scholar
  27. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)
    Article CAS Google Scholar
  28. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)
    Article CAS Google Scholar
  29. Schmid, M. F. et al. A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions. PLoS Pathog. 8, e1002961 (2012)
    Article CAS Google Scholar
  30. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)
    Article CAS Google Scholar
  31. Henderson, R. et al. Outcome of the first electron microscopy validation task force meeting. Structure 20, 205–214 (2012)
    Article CAS Google Scholar
  32. Baker, M. L. et al. Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling. Proc. Natl Acad. Sci. USA 110, 12301–12306 (2013)
    Article ADS CAS Google Scholar
  33. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)
    Article CAS Google Scholar

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Acknowledgements

This research was supported by grants from the Robert Welch Foundation (Q1242) and National Institutes of Health (P41GM123832 to W.C.; AI0175208 and PN2EY016525 to W.C. and J.A.K.; GM080139 to S.J.L.; T15LM007093 through the Gulf Coast Consortia to W.D. and R.H.R.; T32GM007330 through the MSTP to R.H.R.). We thank J. G. Galaz-Montoya and R. N. Irobalieva for editing of the manuscript.

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Author notes

  1. John Flanagan
    Present address: Present address: FEI, 5350 Dawson Creek Drive, Hillsboro, Oregon 97124, USA.,

Authors and Affiliations

  1. Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, 77030, Texas, USA
    Wei Dai, Caroline Fu, John Flanagan, Htet A. Khant, Xiangan Liu, Ryan H. Rochat, Steve J. Ludtke, Michael F. Schmid & Wah Chiu
  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA
    Desislava Raytcheva, Cameron Haase-Pettingell & Jonathan A. King
  3. Department of Biology, Northeastern University, Boston, 02115, Massachusetts, USA
    Desislava Raytcheva & Jacqueline Piret
  4. Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, 77030, Texas, USA
    Ryan H. Rochat, Steve J. Ludtke, Michael F. Schmid & Wah Chiu
  5. National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan ,
    Kuniaki Nagayama

Authors

  1. Wei Dai
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  2. Caroline Fu
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  3. Desislava Raytcheva
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  4. John Flanagan
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  5. Htet A. Khant
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  6. Xiangan Liu
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  7. Ryan H. Rochat
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  8. Cameron Haase-Pettingell
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  9. Jacqueline Piret
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  10. Steve J. Ludtke
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  11. Kuniaki Nagayama
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  12. Michael F. Schmid
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  13. Jonathan A. King
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  14. Wah Chiu
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Contributions

W.D., D.R. and C.F. prepared the samples and conducted the infection experiments under the advice of C.H.-P. and J.P. W.D. collected the image data and reconstructed the tomograms; C.F. and H.A.K. established the Zernike phase plate imaging conditions in the microscope; K.N. provided the phase plates for imaging; R.H.R. performed the statistical analysis. W.D. and M.F.S. developed the imaging processing methods and solved the structures of the phage assembly intermediates; W.D. and X.L. refined the structures; J.F. and S.J.L. developed the symmetry-search algorithm for subvolume alignment; W.D., M.F.S., J.A.K. and W.C. interpreted the structures and wrote the manuscript.

Corresponding author

Correspondence toWah Chiu.

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Extended data figures and tables

Supplementary information

Zernike phase contrast tilt series images of a Syn5-infected WH8109 cell at an intermediate stage of infection

Tilt series of a frozen, hydrated Syn5-infected WH8109 cell was collected manually with an electron energy of 200 kV under low dose conditions on a 4kx4k CCD camera at 25,000× microscope magnification. The tilt angles ranged from -60° to 60° at 3° step increments. The accumulated electron exposure for the specimen in this tilt series was 40-50 electrons/Å2. The sampling of the data was calibrated to be 4.52Å/pixel. (MOV 11476 kb)

Volume rendering and annotation of the tomogram shown in video S1 with colour representations as in Fig. 2c

The tomogram was reconstructed using IMOD software. It is displayed both section-by-section and by volume rendering. Features are annotated by colours to designate molecular components attached and inside the cells, including cell envelope, thylakoid membrane, carboxysome, P-granules, ribosomes, infecting phages and phage assembly intermediates. (MOV 27885 kb)

Zernike phase contrast tilt series images of a Syn5-infected WH8109 cell at a late stage of infection with a ruptured cell membrane

Tilt series of frozen, hydrated Syn5-infected WH8109 cell was collected manually with an electron energy of 200 kV under low dose conditions on a 4kx4k CCD camera at 25,000× microscope magnification. The tilt angles ranged from -60° to 60° at 3° step increments. The accumulated electron exposure for the specimen in this tilt series was 40-50 electrons/Å2. The sampling of the data was calibrated to be 4.52Å/pixel. (MOV 13083 kb)

Volume rendering and annotation of the tomogram shown in video S3 with colour representations as in Fig. 1

The tomogram was reconstructed using IMOD software. It is displayed both section-by-section and by volume rendering. Features are annotated by colours to designate molecular components attached and inside the cells, including cell envelope, thylakoid membrane, carboxysome, P-granules, ribosomes, infecting phages and phage assembly intermediates. An increased number of DNA containing phage particles are visible, and overall contrast is enhanced over that in video S2, because the cell is beginning to rupture at this late stage of infection. (MOV 28142 kb)

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Dai, W., Fu, C., Raytcheva, D. et al. Visualizing virus assembly intermediates inside marine cyanobacteria.Nature 502, 707–710 (2013). https://doi.org/10.1038/nature12604

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Editorial Summary

High-resolution cryo-electron tomography

Wah Chiu and colleagues report the first application of Zernike phase contrast (ZPC) electron cryo-tomography to examine cellular processes without the need for labelling or sectioning. They use this technique to visualize the maturation of the cyanophage Syn5 inside its host cell, identifying subcellular compartments and distinct Syn5 assembly intermediates.

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