key: cord-301942-ppa7gb95 authors: Neuman, Benjamin W.; Adair, Brian D.; Yoshioka, Craig; Quispe, Joel D.; Milligan, Ronald A.; Yeager, Mark; Buchmeier, Michael J. title: Ultrastructure of SARS-CoV, FIPV, and MHV Revealed by Electron Cryomicroscopy date: 2006 journal: The Nidoviruses DOI: 10.1007/978-0-387-33012-9_31 sha: doc_id: 301942 cord_uid: ppa7gb95 nan * The Scripps Research Institute, La Jolla, California. The current understanding of coronavirus ultrastructure relies heavily on transmission electron microscopy of negatively stained images. Such images typically show desiccated specimens and derive contrast from the accumulation of heavy metal negative stains, distorting the sample in the resulting image. Electron cryomicroscopy (cryo-EM) avoids some of the drawbacks of negative staining by imaging frozen specimens preserved in a fully hydrated state in vitreous ice. Cryo-EM images typically derive contrast solely from the density of the imaged sample and the surrounding ice matrix. A limited analysis of porcine transmissible gastroenteritis virus (TGEV) imaged by cryo-EM has been previously reported. 1 In this report we present a more detailed description of the supramolecular design of three coronaviruses: SARS coronavirus (SARS-CoV), feline infectious peritonitis virus (FIPV), and murine hepatitis virus (MHV). Coronaviruses are usually classified as non-icosahedral, pleomorphic, enveloped viruses. Cryo-EM has revealed that other pleomorphic viruses have a roughly spherical appearance, studded with projections that correspond to oligomers of the attachment and fusion protein. Examples include influenza virus [2] [3] [4] ; several retroviruses such as foamy virus, 5 human immunodeficiency virus, 6-10 murine leukemia virus, 11 and Rous sarcoma virus 12, 13 ; La Crosse virus 14, 15 ; Sendai virus 16 ; and Pichinde, Tacaribe, and lymphocytic choriomeningitis viruses. 17 Based on single-particle image analysis of arenaviruses imaged by cryo-EM, we have proposed that pleomorphic arenavirus particles are constructed from overlapping paracrystalline lattices of proteins, and that these lattices span the viral membrane. 11 We hypothesized that coronaviruses may contain a similar supramolecular arrangement of proteins comprising a membrane-proximal scaffold. Here we used cryo-EM to examine the ultrastructure of a selection of coronaviruses representing two of the three proposed phylogenetic groups. Particles of SARS-CoV, FIPV, and MHV were prepared from Vero-E6, AK-D, and DBT cells, respectively. MHV and SARS-CoV were also produced in cells cultured with tunicamycin, to form spike-depleted particles with low infectivity. For safety reasons, all particles were fixed with 10% (for SARS-CoV) or 1% (for FIPV and MHV) formalin in pH 6.5 HEPES-buffered physiological saline before imaging. All viruses were collected by sucrose gradient ultracentrifugation, and each remained highly infectious until fixed. Each virus appeared approximately round in cryo-EM images, with a fringe of spikes protruding from the viral membrane and a region of lower density near the virion center ( Fig. 1A-B) . The average diameter of the membrane-enclosed part of each virus was similar, ranging from ~830 Å for SARS-CoV to ~960 Å for FIPV (Fig. 1C) . The diameters of MHV and SARS-CoV virions were distributed more tightly than diameters of FIPV or spike-depleted, tunicamycin-grown MHV. The mean diameters of native and tunicamycin-grown MHV were similar. Particles of SARS-CoV and MHV produced from tunicamycin-treated cells lacked the characteristic fringe of spikes, but were otherwise indistinguishable from particles grown under standard culture conditions (Fig. 2) . Spike-depleted SARS-CoV particles appeared similar to spike-depleted MHV particles in negative stain, but were produced in lower yield, not suitable for effective cryo-EM imaging. Particles were imaged in several degrees of focus in order to emphasize different structural elements. Fine features such as the phospholipids headgroup densities of the viral membrane and individual nucleocapsid protein densities are revealed more clearly in images recorded relatively near to focus (Figs. 1A-B, 2A right) . Images recorded farther from focus reveal spikes more clearly at the edge and center of each particle ( Fig. 2A, left) . Preparations of each virus contained a small amount of material that was consistent with the appearance of coronavirus ribonucleoprotein (RNP). 18 A particularly interesting image of a SARS-CoV particle trapped in a partially uncoated state at the time of freezing (Fig. 3A-B) shows the spiral RNP partially uncoiled from an approximately round RNP core. The RNP proximal to the extruded membrane segment remains roughly spherical, and appears to be connected to the inner face of the membrane at the ruptured fringe (Fig. 3C ). Each virus is covered with spikes that extend ~200 Å from the peak density of the headgroups in the outer leaflet of the viral membrane. There appears to be a gap between adjacent end-projected spikes near the virion center ( Fig. 2A right, for example) . The arrangement of spike densities near the center of some particles approximates a rhombus, which would not be inconsistent with a paracrystalline organization of spikes as observed in the virions of pleomorphic arenavirus particles, 17 or a local hexagonal close-packing of structural proteins as observed in retroviral particles. 11 Coronavirus particles, as previously pleomorphic virions we have examined by cryo-EM. The observed variability in shape and size of the coronavirus particle would typically be considered inconsistent with icosahedral organization. The observation that the helical RNP is retained in a rough sphere through apparent interaction with proteins resident in the viral membrane is consistent with the spherical arrangement of the viral nucleocapsid proposed for TGEV. 19 However, further image analysis and biochemical experimentation will be required to determine the supramolecular organization of the virion. Some of the work described here was conducted at the National Resource for Automated Molecular Microscopy (NRAMM), which is supported by the National Institutes of Health though the National Center for Research Resources' P41 program (RR17573). This work was supported by NIH grants AI059799, AI025913, and NS41219, and by NIH/NIAID contract HHSN266200400058C. The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins Electron microscopy of influenza virus. 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