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1、Cryo-electron tomography of mouse hepatitis virus: Insights into the structure of the coronavirion Montserrat Ba rcenaa, Gert T. Oostergetelb, Willem Bartelinkc, Frank G. A. Faasa, Arie Verkleijd, Peter J. M. Rottierc, Abraham J. Kostera,1, and Berend Jan Boschc aDepartment of Molecular Cell Biology
2、, Electron Microscopy Section, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands;bBiophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9497 AG, Groningen, The Netherlands;cDepartment of Infectious Diseases and Immunology, Virol
3、ogy Division, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands; anddDepartment of Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands Edited by Stanley Perlman, University of Iowa, Iowa City, IA, and accepted by the Editorial Board Novem
4、ber 20, 2008 (received for review June 3, 2008) Coronaviruses are enveloped viruses containing the largest re- ported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we rep
5、ort the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size (?85 nm envelope diameter). The viral envelope ex
6、hibits an unusual thickness (7.8 ? 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled struc- tures and tubul
7、ar shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be exten- sively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of ?4 nm.
8、 Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confi rm all the general features and global architecture observed for MHV. We propose a general model for the structu
9、re of the coronavirion in which our own and published observations are combined. coronaviruses ? enveloped viruses ? plus-stranded RNA viruses ? transmissible gastroenteritis coronavirus C oronaviruses (CoVs) are large, enveloped, plus-stranded RNA viruses that infect a wide variety of avians and ma
10、mmals. Several CoVs are human pathogens, including the causative agent of severe acute respiratory syndrome (SARS) (1, 2). The SARS outbreak in 20022003 served as a grim reminder of these viruses potential to cross the species barrier and triggered a renewed interest in CoVs. Since then, new CoVs ha
11、ve come to expand the list of discovered members, with two more respiratory human CoVs among them (3, 4). The current virion model of CoVs, based on morphological and biochemical data, depicts a pleomorphic particle that is roughly spherical but shows variations in size (80120 nm) and shape (reviewe
12、d in refs. 5, 6). The virion contains the ribonu- cleoprotein (RNP) core, consisting of a genomic RNA that is the largest among all RNA viruses (?30 kb), in complex with the nucleocapsid (N) protein (?380450 aa). The RNP is sur- rounded by a lipidic envelope. Three viral proteins are anchored in the
13、 envelope of all CoVs: the abundant triple-spanning membrane (M) protein (?230 aa), the envelope (E) protein (?100 aa; present in only small amounts), and the spike (S) protein (?1,300 aa), forming trimeric spikes. The spikes are responsible for receptor binding and membrane fusion and extend radial
14、ly from the envelope, giving the virions a solar image to which coronaviruses owe their name. The M protein appears to be the building brick of the virion and is thought to orchestrate virion assembly via interactions with all viral com- ponents, including itself. Virus-like particles devoid of RNP
15、and S can be formed upon co-expression of M and E (7). Despite multiple biochemical and morphological studies, many fundamental aspects of the virion architecture remain to be elucidated. The structure of the coronavirus RNP core has been particularly elusive. In negatively stained intact virions, t
16、he interior is not visible, and thus early studies focused on samples in which the viral envelope was disrupted and the inner content released. RNPs extracted in this way from several CoVs appear to have helical symmetry (810). However, under certain con- ditions, intermediate spherical nucleocapsids have been re- ported (11, 12). These structures, which showed polygonal profiles, were devoid of S protein and lipids but, in addition to RNA and the N protein, contained the M protein (12). These a
