scholarly journals Structural Dynamics of Nonenveloped Virus Disassembly Intermediates

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Kimi Azad ◽  
Manidipa Banerjee
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Icosahedral Symmetry ◽  
Fold Axis ◽  
Three Dimensional Reconstruction ◽  
Cryo Electron Microscopy ◽  
Successful Infection ◽  
Dimensional Reconstruction ◽  
Nonenveloped Virus

ABSTRACT The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive understanding of how stable, uniform icosahedrons disassemble remains elusive, mainly due to the complexities involved in isolating transient intermediates. We utilized incremental heating to systematically characterize the disassembly pathway of a model nonenveloped virus and identified an intriguing link between virus maturation and disassembly. Further, we isolated and characterized two intermediates by cryo-electron microscopy and three-dimensional reconstruction, without imposing icosahedral symmetry. The first intermediate displayed a series of major, asymmetric alterations, whereas the second showed that the act of genome release, through the 2-fold axis, is actually confined to a small section on the capsid. Our study thus presents a comprehensive structural analysis of nonenveloped virus disassembly and emphasizes the asymmetric nature of programmed conformational changes. IMPORTANCE Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by nonenveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates: “eluted particles” and “puffed particles” of an insect nodavirus, Flock House virus (FHV). Cryo-electron microscopy and three-dimensional reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.

Organized packing of RNA inside viruses as revealed by cryo-electron microscopy and x-ray diffraction analysis

1992 ◽  
Vol 50 (1) ◽  
pp. 454-455
Author(s):  
T. S. Baker ◽  
R. H. Cheng ◽  
J. E. Johnson ◽  
N. H. Olson ◽  
G. J. Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Diffraction Analysis ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
Three Dimensional Reconstruction ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Dimensional Reconstruction ◽  
Microscopy Techniques

The development of modern electron microscopy techniques to visualize the hydrated structures of biological macromolecules has stimulated many new studies, especially with viruses and virus-macromolecule complexes that are too large to study with current x-ray crystallographic methods, Cryo-electron microscopy (cryoEM) in conjunction with three-dimensional reconstruction procedures is capable of revealing both external and internal features of these structures.

scholarly journals Catching a Virus in the Act of RNA Release: a Novel Poliovirus Uncoating Intermediate Characterized by Cryo-Electron Microscopy

2010 ◽  
Vol 84 (9) ◽  
pp. 4426-4441 ◽  
Author(s):  
Hazel C. Levy ◽  
Mihnea Bostina ◽  
David J. Filman ◽  
James M. Hogle
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Cell Entry ◽  
Continuous Series ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Reconstruction Methods ◽  
Dimensional Classification ◽  
Dimensional Reconstruction

ABSTRACT Poliovirus infection requires that the particle undergo a series of conformational transitions that lead to cell entry and genome release. In an effort to understand the conformational changes associated with the release of the RNA genome, we have used cryo-electron microscopy to characterize the structure of the 80S “empty” particles of poliovirus that are thought to represent the final product of the cell entry pathway. Using two-dimensional classification methods, we show that preparations of 80S particles contain at least two structures, which might represent snapshots from a continuous series of conformers. Using three-dimensional reconstruction methods, we have solved the structure of two distinct forms at subnanometric resolution, and we have built and refined pseudoatomic models into the reconstructions. The reconstructions and the derived models demonstrate that the two structural forms are both slightly expanded, resulting in partial disruption of interprotomer interfaces near their particle 2-fold axes, which may represent the site where RNA is released. The models demonstrate that each of the two 80S structures has undergone a unique set of movements of the capsid proteins, associated with rearrangement of flexible loops and amino-terminal extensions that participate in contacts between protomers, between pentamers, and with the viral RNA.

Three-Dimensional Reconstruction of Native Androctonus Australis Hemocyanin

1990 ◽  
Vol 48 (1) ◽  
pp. 452-453
Author(s):  
Nicolas Boisset ◽  
Jean-Christophe Taveau ◽  
Jean Lamy ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Solid Body ◽  
Body Surface ◽  
Three Dimensional ◽  
Staining Technique ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction ◽  
View Reconstruction ◽  
Surface Representations ◽  
Top View

Hemocyanin, the respiratory pigment of the scorpion Androctonus australis is composed of 24 kidney shaped subunits. A model of architecture supported by many indirect arguments has been deduced from electron microscopy (EM) and immuno-EM. To ascertain, the disposition of the subunits within the oligomer, the 24mer was submitted to three-dimensional reconstruction by the method of single-exposure random-conical tilt series.A sample of native hemocyanin, prepared with the double layer negative staining technique, was observed by transmisson electron microscopy under low-dose conditions. Six 3D-reconstructions were carried out indenpendently from top, side and 45°views. The results are composed of solid-body surface representations, and slices extracted from the reconstruction volume.The main two characters of the molecule previously reported by Van Heel and Frank, were constantly found in the solid-body surface representations. These features are the presence of two different faces called flip and flop and a rocking of the molecule around an axis passing through diagonnally opposed hexamers. Furthermore, in the solid-body surface of the top view reconstruction, the positions and orientations of the bridges connecting the half molecules were found in excellent agreement with those predicted by the model.

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

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