Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction (original) (raw)
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Journal of General Virology, 1986
The inner capsid structure of the OSU strain of porcine rotavirus was studied by electron microscopy of freeze-dried preparations and of negatively stained chemically disrupted virus particles. The analysis of the particles by the freeze-drying technique revealed a T : 13 1 (laevo) symmetry for the organization of the inner capsid. Treatment of single-capsid rotavirus particles with 30~ formamide or 5 M-urea resulted in their degradation, giving rise to very similar products, corresponding to isolated vertices, edges and faces of the virus icosahedron. An analysis of such structures confirmed the triangulation number and handedness of the rotavirus inner capsid, and provided evidence for the open-mesh model, in which the five-and six-coordinated axes are represented by 'holes" formed by smaller trimeric morphological subunits.
Three dimensional morphology of rabies virus studied by cryo-electron tomography
Journal of Structural Biology, 2011
The rabies virus (RABV) continues to be a worldwide health problem. RABV contains a single-stranded RNA genome that associates with the nucleoprotein N. The resulting ribonucleoprotein complex is surrounded by matrix protein M, lipid bilayer and glycoprotein G. RABV was reported to organize in bullet-like virions, but the role of each viral component in adopting this morphology is unclear. We present here a cryo-electron tomography study of RABV showing additional morphologies consisting in bullet-like virions containing a tubular, lipidic appendage having G-protein at its apex. In addition, there was evidence for an important fraction of pleomorphic particles. These pleomorphic forms differed in the amount of membrane-associated M-, M/N-protein providing interesting insight into its role in viral morphogenesis. In the absence of membrane-associated M-, M/N-protein viral morphology was almost spherical. Other images, showing straight membrane portions, correlate with the M-protein recruitment at the membrane independently of the presence of the G-protein. The viral membrane was found to contain a negative net charge indicating that M-, M/N-protein-membrane charge attraction drives this interaction.
Journal of General Virology, 1985
The major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 M-CaCI~ removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomerie form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.
Rotavirus Architecture at Subnanometer Resolution
Journal of Virology, 2008
Rotavirus, a nonturreted member of the Reoviridae , is the causative agent of severe infantile diarrhea. The double-stranded RNA genome encodes six structural proteins that make up the triple-layer particle. X-ray crystallography has elucidated the structure of one of these capsid proteins, VP6, and two domains from VP4, the spike protein. Complementing this work, electron cryomicroscopy (cryoEM) has provided relatively low-resolution structures for the triple-layer capsid in several biochemical states. However, a complete, high-resolution structural model of rotavirus remains unresolved. Combining new structural analysis techniques with the subnanometer-resolution cryoEM structure of rotavirus, we now provide a more detailed structural model for the major capsid proteins and their interactions within the triple-layer particle. Through a series of intersubunit interactions, the spike protein (VP4) adopts a dimeric appearance above the capsid surface, while forming a trimeric base an...
Electron cryo-tomographic structure of cystovirus ?12
Virology, 2008
Bacteriophage ϕ12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species ϕ6, host infection requires binding of a multimeric P3 complex to type IV pili. In species ϕ8, ϕ12, and ϕ13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In ϕ8 the protein components are designated P3a and P3b while in species ϕ12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T = 13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact ϕ12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.
Electron cryo-tomographic structure of cystovirus ϕ12
Virology, 2008
Bacteriophage ϕ12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species ϕ6, host infection requires binding of a multimeric P3 complex to type IV pili. In species ϕ8, ϕ12, and ϕ13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In ϕ8 the protein components are designated P3a and P3b while in species ϕ12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T = 13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact ϕ12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.
Structures of enveloped virions determined by cryogenic electron microscopy and tomography
Complementary Strategies to Understand Virus Structure and Function, 2019
Bundibugyo virus (BDBV) Charge coupled device (CCD) Chikungunya virus (CHIKV) Contrast transfer function (CTF) Cryogenic electron microscopy (cryo-EM) Dengue virus (DENV) Deoxyribonucleic acid (DNA) Direct electron detector (DED) Ebola virus (EBOV) Eastern equine encephalitis virus (EEEV) Endosomal sorting complex required for transport (ESCRT) Enterovirus 71 (EV71) Focused ion beam (FIB) Glycoprotein (GP) Hazara virus (HAZV) Hemagglutinin (HA) Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Human parainfluenza virus 3 (HPIV3) Japanese encephalitis virus (JEV) Lassa virus (LASV) Major capsid protein (MCP) Marburg virus (MARV) Measles virus (MeV) Mouse hepatitis virus (MHV) Mucin-like domain (MLD) Vesicular stomatitis virus (VSV) Virus-like particle (VLP) Volta phase plate (VPP) West Nile virus (WNV) Zika virus (ZIKV)
Cryo-electron tomography of vaccinia virus
Proceedings of the National Academy of Sciences, 2005
The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4 -6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brickshaped viral particles with slightly rounded edges and dimensions of Ϸ360 ؋ 270 ؋ 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of Ϸ18 -19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.