The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.

Assistance Publique des Hôpitaux de Marseille La Timone 13005 Marseille France

Institute of Physics AS CR v v i Na Slovance 2 18221 Prague 8 Czech Republic

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Husargatan 3 SE 75124 Uppsala Sweden

National Institute for Physiological Sciences Okazaki Aichi 444 8585 Japan

Structural and Genomic Information Laboratory UMR 7256 Centre National de la Recherche Scientifique and Aix Marseille University Marseille 13288 France

Zobrazit více v PubMed

La Scola B, et al. A giant virus in amoebae. Science. 2003;299:2033. doi: 10.1126/science.1081867. PubMed DOI

Abergel C, Legendre M, Claverie JM. The rapidly expanding universe of giant viruses: Mimivirus, Pandoravirus, Pithovirus and Mollivirus. FEMS microbiology reviews. 2015;39:779–796. doi: 10.1093/femsre/fuv037. PubMed DOI

Raoult D, et al. The 1.2-megabase genome sequence of Mimivirus. Science. 2004;306:1344–1350. doi: 10.1126/science.1101485. PubMed DOI

Philippe N, et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science. 2013;341:281–286. doi: 10.1126/science.1239181. PubMed DOI

Scheid P, Balczun C, Schaub GA. Some secrets are revealed: parasitic keratitis amoebae as vectors of the scarcely described pandoraviruses to humans. Parasitology research. 2014;113:3759–3764. doi: 10.1007/s00436-014-4041-3. PubMed DOI

Antwerpen, M. H. et al. Whole-genome sequencing of a pandoravirus isolated from keratitis-inducing acanthamoeba. Genome announcements3, 10.1128/genomeA.00136-15 (2015). PubMed PMC

Legendre M, et al. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proceedings of the National Academy of Sciences of the United States of America. 2014;111:4274–4279. doi: 10.1073/pnas.1320670111. PubMed DOI PMC

Levasseur A, et al. Comparison of a Modern and Fossil Pithovirus Reveals Its Genetic Conservation and Evolution. Genome biology and evolution. 2016;8:2333–2339. doi: 10.1093/gbe/evw153. PubMed DOI PMC

Andreani, J. et al. Cedratvirus, a Double-Cork Structured Giant Virus, is a Distant Relative of Pithoviruses. Viruses8, doi:10.3390/v8110300 (2016). PubMed PMC

Legendre M, et al. In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba. Proceedings of the National Academy of Sciences of the United States of America. 2015;112:E5327–5335. doi: 10.1073/pnas.1510795112. PubMed DOI PMC

Zauberman N, et al. Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. PLoS biology. 2008;6:e114. doi: 10.1371/journal.pbio.0060114. PubMed DOI PMC

Xiao C, et al. Cryo-electron microscopy of the giant Mimivirus. Journal of molecular biology. 2005;353:493–496. doi: 10.1016/j.jmb.2005.08.060. PubMed DOI

Xiao C, et al. Structural studies of the giant mimivirus. PLoS biology. 2009;7:e92. doi: 10.1371/journal.pbio.1000092. PubMed DOI PMC

Claverie JM, Abergel C. Mimivirus and its virophage. Annual review of genetics. 2009;43:49–66. doi: 10.1146/annurev-genet-102108-134255. PubMed DOI

Piacente F, et al. Giant DNA virus mimivirus encodes pathway for biosynthesis of unusual sugar 4-amino-4,6-dideoxy-D-glucose (Viosamine) The Journal of biological chemistry. 2012;287:3009–3018. doi: 10.1074/jbc.M111.314559. PubMed DOI PMC

Piacente F, et al. Characterization of a UDP-N-acetylglucosamine biosynthetic pathway encoded by the giant DNA virus Mimivirus. Glycobiology. 2014;24:51–61. doi: 10.1093/glycob/cwt089. PubMed DOI

Mutsafi Y, Shimoni E, Shimon A, Minsky A. Membrane assembly during the infection cycle of the giant Mimivirus. PLoS pathogens. 2013;9:e1003367. doi: 10.1371/journal.ppat.1003367. PubMed DOI PMC

Ekeberg T, et al. Three-dimensional reconstruction of the giant mimivirus particle with an x-ray free-electron laser. Physical review letters. 2015;114:098102. doi: 10.1103/PhysRevLett.114.098102. PubMed DOI

Arslan D, Legendre M, Seltzer V, Abergel C, Claverie JM. Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:17486–17491. doi: 10.1073/pnas.1110889108. PubMed DOI PMC

Yoosuf N, et al. Related giant viruses in distant locations and different habitats: Acanthamoeba polyphaga moumouvirus represents a third lineage of the Mimiviridae that is close to the megavirus lineage. Genome biology and evolution. 2012;4:1324–1330. doi: 10.1093/gbe/evs109. PubMed DOI PMC

Grimm R, Typke D, Barmann M, Baumeister W. Determination of the inelastic mean free path in ice by examination of tilted vesicles and automated most probable loss imaging. Ultramicroscopy. 1996;63:169–179. doi: 10.1016/0304-3991(96)00035-6. PubMed DOI

Koster AJ, et al. Perspectives of molecular and cellular electron tomography. Journal of structural biology. 1997;120:276–308. doi: 10.1006/jsbi.1997.3933. PubMed DOI

Murata K, et al. Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography. Ultramicroscopy. 2014;146:39–45. doi: 10.1016/j.ultramic.2014.05.008. PubMed DOI

Danev R, Nagayama K. Phase plates for transmission electron microscopy. Methods in enzymology. 2010;481:343–369. doi: 10.1016/S0076-6879(10)81014-6. PubMed DOI

Murata K, et al. Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure. 2010;18:903–912. doi: 10.1016/j.str.2010.06.006. PubMed DOI PMC

Rochat RH, et al. Seeing the portal in herpes simplex virus type 1 B capsids. Journal of virology. 2011;85:1871–1874. doi: 10.1128/JVI.01663-10. PubMed DOI PMC

Dai W, et al. Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages. Nature protocols. 2014;9:2630–2642. doi: 10.1038/nprot.2014.176. PubMed DOI PMC

Dai W, et al. Visualizing virus assembly intermediates inside marine cyanobacteria. Nature. 2013;502:707–710. doi: 10.1038/nature12604. PubMed DOI PMC

Malac M, Beleggia M, Kawasaki M, Li P, Egerton RF. Convenient contrast enhancement by a hole-free phase plate. Ultramicroscopy. 2012;118:77–89. doi: 10.1016/j.ultramic.2012.02.004. PubMed DOI

Hosogi N, Sen A, Iijima H. Comparison of cryo TEM images obtained with Zernike and Hole-Free Phase Plates. Microsc. Microanal. 2015;21:1389–1390. doi: 10.1017/S1431927615007722. DOI

Danev R, Buijsse B, Khoshouei M, Plitzko JM, Baumeister W. Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proceedings of the National Academy of Sciences of the United States of America. 2014;111:15635–15640. doi: 10.1073/pnas.1418377111. PubMed DOI PMC

Grunewald K, et al. Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science. 2003;302:1396–1398. doi: 10.1126/science.1090284. PubMed DOI

Hayashida M, Malac M. Practical electron tomography guide: Recent progress and future opportunities. Micron. 2016;91:49–74. doi: 10.1016/j.micron.2016.09.010. PubMed DOI

Rez P, Larsen T, Elbaum M. Exploring the theoretical basis and limitations of cryo-STEM tomography for thick biological specimens. Journal of structural biology. 2016;196:466–478. doi: 10.1016/j.jsb.2016.09.014. PubMed DOI

Wolf SG, Houben L, Elbaum M. Cryo-scanning transmission electron tomography of vitrified cells. Nature methods. 2014;11:423–428. doi: 10.1038/nmeth.2842. PubMed DOI

Aoyama, K., Nagano, K. & Mitsuoka, K. Optimization of STEM imaging conditions for cryo-tomography. Microscopy, 1-5, 10.1093/jmicro/dfx003 (2017). PubMed

Marko M, Hsieh C, Schalek R, Frank J, Mannella C. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nature methods. 2007;4:215–217. doi: 10.1038/nmeth1014. PubMed DOI

Murata K, Hagiwara S, Kimori Y, Kaneko Y. Ultrastructure of compacted DNA in cyanobacteria by high-voltage cryo-electron tomography. Scientific reports. 2016;6:34934. doi: 10.1038/srep34934. PubMed DOI PMC

Satoh K, et al. Effective synaptome analysis of itch-mediating neurons in the spinal cord: A novel immunohistochemical methodology using high-voltage electron microscopy. Neuroscience letters. 2015;599:86–91. doi: 10.1016/j.neulet.2015.05.031. PubMed DOI

Satoh K, et al. Three-dimensional visualization of multiple synapses in thick sections using high-voltage electron microscopy in the rat spinal cord. Data in brief. 2015;4:566–570. doi: 10.1016/j.dib.2015.07.005. PubMed DOI PMC

Malis T, Cheng SC, Egerton RF. EELS log-ration technique for specimen-thickness measurment in the TEM. J Electron Microsc Tech. 1988;8:193–200. doi: 10.1002/jemt.1060080206. PubMed DOI

Sun S, Shi S, Leapman R. Water distributions of hydrated biological specimens by valence electron energy loss spectroscopy. Ultramicroscopy. 1993;50:127–139. doi: 10.1016/0304-3991(93)90003-G. PubMed DOI

Dohnalkova AC, et al. Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy. Applied and environmental microbiology. 2011;77:1254–1262. doi: 10.1128/AEM.02001-10. PubMed DOI PMC

Zhang X, et al. Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:14837–14842. doi: 10.1073/pnas.1107847108. PubMed DOI PMC

Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. Journal of structural biology. 1996;116:71–76. doi: 10.1006/jsbi.1996.0013. PubMed DOI