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Visualizing virus assembly intermediates inside marine cyanobacteria

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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Figure 1: ZPC cryoET enables direct recognition of cellular components of the Syn5-infected WH8109 cells.
Figure 2: ZPC cryoET of WH8109 cells before and after infection with Syn5 phage.
Figure 3: Phage progeny average maps reveal diverse assembly intermediates during phage assembly.
Figure 4: Phage assembly model revealed by ZPC cryoET.

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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.

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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

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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 to Wah Chiu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 ZPC improves contrast of cryoET images and reveals detailed structural features of Syn5-infected cells.

a, A conventional EM image of a Syn5-infected WH8109 cell. b, A ZPC image of the same cell as shown in a under the same imaging conditions.

Extended Data Figure 2 ZPC-cryoEM single-particle images of biochemically purified mature Syn5 phage.

The particles are shown with the tail pointing down and the wavy horn pointing up. The tail fibres appear to have variable conformations.

Extended Data Figure 3 General linear modelling of cellular carboxysome number with progression of infection.

The number of carboxysomes remains roughly constant as infection progresses, indicating that their variation does not correlate with progression of infection. The solid line indicates linear regression; the dashed lines indicate the 95% confidence limits.

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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