Cryo-electron Microscopy of the Giant Mimivirus

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2 doi: /j.jmb J. Mol. Biol. (2005) 353, COMMUNICATION Cryo-electron Microscopy of the Giant Mimivirus Chuan Xiao 1, Paul R. Chipman 1, Anthony J. Battisti 1 Valorie D. Bowman 1, Patricia Renesto 2, Didier Raoult 2 and Michael G. Rossmann 1 * 1 Department of Biological Sciences, Purdue University 915 W. State Street, West Lafayette, IN USA 2 Unité des Rickettsies, Faculté de Médecine, CNRS UMR 6020 IFR 48, 27, bd Jean Moulin Marseille, cedex 5, France *Corresponding author Mimivirus is the largest known virus. Using cryo-electron microscopy, the virus was shown to be icosahedral, covered by long fibers, and appears to have at least two lipid membranes within its protein capsid. A unique vertex, presumably for attachment and infection of the host, can be seen for particles that have a suitable orientation on the micrographs. q 2005 Elsevier Ltd. All rights reserved. Keywords: Mimivirus; cryoem; structure; nucleocytoplasmic dsdna; unique vertex Mimivirus was first isolated from amoebae growing in a British water tower during a 1992 investigation into an outbreak of pneumonia. 1 Recently, Mimivirus DNA and antibodies against Mimivirus were found in patients with community or hospital-acquired pneumonia. 2 The diameter and genome of a Mimivirus is larger than some small bacteria, 1 and more than three times the size of the largest virus previously studied by cryo-electron microscopy (cryoem). 3,4 The nucleocytoplasmic, large, dsdna Mimivirus is the only member of a newly suggested Mimiviridae family. 1 The 1.2 Mbp genome provides sufficient information to allow the virus to perform most of the functions of living cells. 5 The complexity and magnitude of the Mimivirus genome, combined with the large size of the virus, calls into question some of the established divisions between viruses and singlecell organisms, as well as their evolution. 5 The largest viruses studied to date with cryoem are the icosahedral Phaeocystis pouchetii virus (PpV01; 2200 Å diameter with a pseudo-triangulation number TZ219), 3 Paramecium bursaria Chlorella virus type 1 (PBCV-1; 1900 Å diameter, pseudo- C.X. and P.R.C. contributed equally to this work. Abbreviations used: cryoem, cryo-electron microscopy; PBCV-1, Paramecium bursaria Chlorella virus type 1; CCD, charge coupled device; CTF, contrast transfer function. address of the corresponding author: mr@purdue.edu triangulation number TZ169), and Chilo iridescent virus (CIV, 1850 Å diameter, pseudo-triangulation number TZ147). 4 The crystal structure of the PBCV-1 major capsid protein, Vp54, was determined to 2.0 Å resolution by X-ray crystallography and fitted into a three-dimensional cryoem map of the PBCV-1 virion. 6,7 This protein, like the major capsid proteins of CIV, adenovirus, 8 and bacteriophage PRD1, 9 has two domains, each being folded into a jelly-roll structure. 6,10,11 Trimers of Vp54 have pseudohexagonal symmetry and form the capsomers that assemble into trisymmetrons around 3-fold axes and pentasymmetrons around 5-fold axes of the viral capsid. 6,12 The capsomers within one trisymmetron are all oriented the same way, but are rotated by about 608 relative to the capsomers in neighboring trisymmetrons, 6,7 creating cleavage lines that become apparent under denaturing conditions. 12 Based on sequence similarity (26.8% sequence identity), it is probable that the major capsid protein (L425) of Mimivirus has a similar structure as Vp54 in PBCV-1 and assembles into a pseudo-hexameric capsomer organized into tri- and pentasymmetrons, as is the case for other large icosahedral dsdna viruses. 10 Whereas L425 is the most abundant capsid protein, there are numerous other proteins in the virion. 5 cryoem requires the sample to be frozen fast enough to produce vitreous ice. Samples frozen in their hydrated state are free of artifacts prevalent in the more traditional methods of EM sample preparations, such as negative staining or thin /$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

3 494 cryoem Structure of Mimivirus sectioning. 13 However, because the vitreous ice adds noise to the image, the thickness of ice is critical. It was a challenge to produce a uniform layer of ice with a thickness equal to or slightly thicker than the enormous diameter of Mimivirus. Initial attempts at freezing the purified sample used a Vitrobot (FEI Company, USA), a range of blotting parameters and several different perforated support films on the EM grids. These attempts yielded very little useable data but did produce several clear images of the large virus, enough to merit further effort. Ultimately, freezing was done manually, with a guillotine style plunging device and Quantifoil S7/2 grids (Quantifoil Micro Tools GmbH, Germany). No other special provisions, such as glow discharging, humidity control or the addition of more carbon to create a thicker support film, were deemed necessary. A lower success rate than is usual for particles with smaller diameters was obtained for embedding Mimivirus in vitreous ice. The film data were recorded at a nominal magnification of 24,000! using a modified film holder, film transport mechanism and film guide plate (P.C. & M. Sherman, unpublished results). These modifications increased the signal-to-noise ratio by eliminating backscattered electrons that were produced when using the original film plates. Films were scanned on a Zeiss PHODIS scanner using a 28 mm step size, corresponding to an Å pixel size on the sample. The charge coupled device (CCD) data were recorded at a nominal magnification of 31,500! and interpolated to the same pixel size as the digitized film data. The cryoem micrographs of Mimivirus showed that they have a capsid with a diameter of w5000 Å, covered by w1250 Å long, closely packed fibers, projecting out from the capsid surface (Figure 1). The appearance of the large number of fibers seen in vitrious ice is significantly different from the twisted fiber remnants seen by negative staining. 1 Based on a large number of open reading frames with collagen triple helix repeats in the viral genome, 5 these fibers might consist of cross-linked glycosylated collagen. The dense, 200 Å thick base of the fibers (Figure 1(b)) might be caused by the cross-linking. The forest of fibers increased the required ice thickness to be at least 7500 Å and further decreased the signal-to-noise ratio for determining the capsid structure by image reconstruction techniques. The capsid itself appeared to have three layers of dense matter (Figure 1(b)), Figure 1. cryoem images of Mimivirus. cryoem images were collected on an FEI CM300 microscope with a field emission gun operated at 300 kv, a liquid nitrogen stage, and a defocus range of 13 mm to20mm. (a) An array of particles separated by the viral fibers. (b) An enlarged view of one particle showing at least two possible lipid layers (straight arrows), the unanchored nuclear capsid (white arrowhead), the protein capsid (between jagged arrows), and the fibers with their 200 Å thick base layer (black arrowhead). (c) Enlarged view of one particle showing a unique vertex whose width is about 600 Å (black arrows). Radial distances from the center of the virus are shown in Å. The scale bars represent 2000 Å.

cryoem Structure of Mimivirus 495 probably representing two successive 40 Å thick lipid membranes separated by 60 Å inside an approximately 70 Å thick protein shell.

4 cryoem Structure of Mimivirus 495 probably representing two successive 40 Å thick lipid membranes separated by 60 Å inside an approximately 70 Å thick protein shell. Similar double lipid membrane layers have been found in some poxviruses and in African swine fever virus (ASFV), 14 another large virus that might have a similar double jelly-roll capsid. 10 However, unlike ASFV and Mimivirus, PpV01, PBCV-1, and CIV have only a single membrane layer inside the capsid. 3,4 An unanchored compartment containing the genome is incarcerated within the three dense layers seen in Mimivirus (Figure 1(b)). A few, suitably oriented Mimivirus particles had a unique protruding vertex (Figure 1(c)) similar to that observed in tailed phages and in PBCV-1 while the latter is infecting a host. 15 Most of the particles looked hexagonal in projection when observed in vitrified ice (Figure 1(a)). This permitted the calculation of a 75 Å resolution, three-dimensional cryoem map (Figure 2) using 3003 individual particles from 806 micrographs collected on film and a CCD camera. Because of the ice thickness and the long fibers of the Mimivirus, large underfocus distances were used (see the legend to Figure 1) in order to obtain enough contrast for orientation and origin determination when recording the cryoem images. Although contrast transfer function (CTF) corrections were applied to the images, this did not benefit the reconstruction significantly, since the current resolution had not yet progressed beyond the first minimum of the CTF at about 50 Å. The orientation and origin of each particle were determined by the polar Fourier transform method 16 using a 64 bit parallelized program. An initial model required for starting the reconstruction process was based on a PBCV-1 cryoem map 4 computed to 200 Å resolution, scaled to the size of Mimivirus. The three-dimensional map reconstruction was calculated using a parallelized version of the EM3DR 17 program. The map resolution was determined by finding where the Fourier shell correlation coefficient fell below The cryoem map showed that the assumption of homogenous particles with icosahedral symmetry was acceptable. It is a remarkable achievement for a capsid of this size with a relatively thin protein shell to have such an accurately assembled symmetrical capsid. The center-to-center distance (l) between capsomers in PBCV-1 and related viruses is about 75 Å. 6 Thus, a map with at least 33 Å resolution will be required to resolve individual capsomers in order to determine the T number accurately. Assuming a similar capsomer size for PBCV-1 and Mimivirus, the number of capsomers and their surface packing arrangements can be estimated from the ratio (L/l), where L is the edge distance between neighboring 5-fold vertices. 12 For Mimivirus, this ratio is 34.3, corresponding to a T number of w1179 where Tz(L/l) 2. Provided the majority of particles are identical within a 33 Å resolution limit and the unique vertex does not cause significant deviation from icosahedral symmetry, it will take at least three times more particle images than have been collected to date in order to resolve individual capsomers. However, distortions around the unique vertex, the thickness of the ice, and the presence of the fibers might slow attainment of the required resolution. Nevertheless, by combining X-ray crystallography with the cryoem studies, 19 it should be possible to study Figure 2. cryoem 3D reconstructions. (a) Surface-shaded three-dimensional image reconstructions, whereas (b) shows a central cross-section slab about 50 Å thick. The left half of (a) and (b) are at a contour level that includes the denser base layer of the fibers whereas the right half of (a) and (b) are at a higher contour level that shows the capsid. The internal unanchored nucleocapsid can be seen in the left side of (b) between 800 Å and 1500 Å radii. Radial distances from the center are shown in Å in (b) and can be compared to those in Figure 1(b). The scale bar represents 2000 Å.

5 496 cryoem Structure of Mimivirus how Mimivirus, which approaches the complexity of a single cell, assembles and infects its host. Acknowledgements We are grateful for helpful discussions with Petr Leiman and to Sharon Wilder, Cheryl Tower and Sheryl Kelly for help in the preparation of the manuscript. The work was supported by NIH grant AI to M.G.R. and an award from the Keck Foundation for the purchase of an FEI CM300 electron microscope. The research work of D.R. was supported by CNRS. References 1. La Scola, B., Audic, S., Robert, C., Jungang, L., de Lamballerie, X., Drancourt, M. et al. (2003). A giant virus in amoebae. Science, 299, La Scola, B. (2005). Mimivirus in pneumonia patients. Emerg. Infect. Dis. 11, Yan, X., Chipman, P. R., Castberg, T., Bratbak, G. & Baker, T. S. (2005). The marine algal virus PpV01 has an icosahedral capsid with TZ219 quasisymmetry. J. Virol. 79, Yan, X., Olson, N. H., Van Etten, J. L., Bergoin, M., Rossmann, M. G. & Baker, T. S. (2000). Structure and assembly of large lipid-containing dsdna viruses. Nature Struct. Biol. 7, Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H. et al. (2004). The 1.2-megabase genome sequence of Mimivirus. Science, 306, Nandhagopal, N., Simpson, A. A., Gurnon, J. R., Yan, X., Baker, T. S., Graves, M. V. et al. (2002). The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus. Proc. Natl Acad. Sci. USA, 99, Simpson, A. A., Nandhagopal, N., Van Etten, J. L. & Rossmann, M. G. (2003). Structural analyses of Phycodnaviridae and Iridoviridae. Acta Crystallog. sect. D, 59, Roberts, M. M., White, J. L., Grutter, M. G. & Burnett, R. M. (1986). Three-dimensional structure of the adenovirus major coat protein hexon. Science, 232, Benson, S. D., Bamford, J. K., Bamford, D. H. & Burnett, R. M. (1999). Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures. Cell, 98, Benson, S. D., Bamford, J. K., Bamford, D. H. & Burnett, R. M. (2004). Does common architecture reveal a viral lineage spanning all three domains of life? Mol. Cell, 16, Rossmann, M. G. & Johnson, J. E. (1989). Icosahedral RNA virus structure. Annu. Rev. Biochem. 58, Wrigley, N. G. (1969). An electron microscope study of the structure of Sericesthis iridescent virus. J. Gen. Virol. 5, Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W. (1984). Cryo-electron microscopy of viruses. Nature, 308, Rouiller, I., Brookes, S. M., Hyatt, A. D., Windsor, M. & Wileman, T. (1998). African swine fever virus is wrapped by the endoplasmic reticulum. J. Virol. 72, Van Etten, J. L., Lane, L. C. & Meints, R. H. (1991). Viruses and virus-like particles of eukaryotic algae. Microbiol. Rev. 55, Baker, T. S. & Cheng, R. H. (1996). A model-based approach for determining orientations of biological macromolecules imaged by cryo-electron microscopy. J. Struct. Biol. 116, Xiao, C., Bator, C. M., Bowman, V. D., Rieder, E., He, Y., Hebert, B. et al. (2001). Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1. J. Virol. 75, Baker, T. S., Olson, N. H. & Fuller, S. D. (1999). Adding the third dimension to virus life cycles: threedimensional reconstruction of icosahedral viruses from cryo-electron micrographs. Microbiol. Mol. Biol. Rev. 63, Rossmann, M. G., Morais, M. C., Leiman, P. G. & Zhang, W. (2005). Combining X-ray crystallography and electron microscopy. Structure (Camb.), 13, Edited by W. Baumeister (Received 8 July 2005; received in revised form 23 August 2005; accepted 25 August 2005) Available online 12 September 2005

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Journal of Structural Biology 170 (010) 109 116 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi A tale of two symmetrons: Rules for