Article

Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy

University of Lausanne, Lausanne, Vaud, Switzerland
Journal of Virology (Impact Factor: 4.44). 04/1994; 68(3):1935-41.
Source: PubMed

ABSTRACT

Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts.

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Available from: Karsten Richter, Apr 14, 2014
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    • "Advances in VACV genomics, proteomics, and genetics have contributed to assigning proteins to specific genes in genome, identifying proteins as constituents of the virus particle, and understanding the role of these proteins in the replicative cycle. Electron microscopy has also played an important role in comprehending several aspects of viral morphogenesis and poxvirus virion structure (Condit et al., 2006; Cyrklaff et al., 2005; Dubochet et al., 1994; Heuser, 2005; Moss, 2013; Pedersen et al., 2000; Roos et al., 1996). Although all of these techniques have played an essential role in understanding the biology of VACV, the fine details of the virion structure have yet to be determined. "
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    ABSTRACT: The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion. Published by Elsevier Inc.
    Preview · Article · Dec 2014 · Virology
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    • "The structural features found for the X-ray reconstructed vaccinia virions are fully consistent with the structure of the vaccinia virus known from electron microscopy using either thin sections (Griffiths et al., 2001a), cryo-electron microscopy projections (Cyrklaff et al., 2007; Dubochet et al., 1994) or tomograms (Cyrklaff et al., 2005). Fig. 5a shows two representative planes from cryo-Xray tomographic virus reconstructions where the inner core is placed off-centre inside the outer envelope. "
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    ABSTRACT: We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.
    Full-text · Article · Dec 2011 · Journal of Structural Biology
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    • "The structural features found for the X-ray reconstructed vaccinia virions are fully consistent with the structure of the vaccinia virus known from electron microscopy using either thin sections (Griffiths et al., 2001a), cryo-electron microscopy projections (Cyrklaff et al., 2007; Dubochet et al., 1994) or tomograms (Cyrklaff et al., 2005). Fig. 5a shows two representative planes from cryo-Xray tomographic virus reconstructions where the inner core is placed off-centre inside the outer envelope. "
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    ABSTRACT: Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250 × 270 × 360 nm3. The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7 nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.
    Full-text · Article · Nov 2009 · Journal of Structural Biology
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