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Electron microscopy is the most widely used method for the examination of structure and morphology of virus particles. This technique, which has been applied to viruses since the 1940s, traditionally relied on staining of purified virus particles (or of sections of infected cells) with an electron-dense material. It can yield quite detailed and often beautiful images (Fig. 1.9; see the Appendix) and provided the first rational basis for classification of viruses.

Table 4.2 Nomenclature used in description of virus structure

Term	Synonym	Definition
Subunit (protein subunit)		Single, folded polypeptide chain
Structural unit	Asymmetric unit	Unit from which capsids or nucleocapsids are built; may comprise one protein subunit or multiple, different protein subunits
Capsid	Coat	The protein shell surrounding the nucleic acid genome
Nucleocapsid	Core	The nucleic acid-protein assembly packaged within the virion; used when this assembly is a discrete substructure of a particle
Envelope	Viral membrane	The host cell-derived lipid bilayer carrying viral glycoproteins
Virion		The infectious virus particle

The greatest contrast between virus particle and stain (negative contrast) occurs where portions of the folded protein chain protrude from the surface. Consequently, surface knobs or projections, termed morphological units, are the main features identified by this method. However, because these surface features are often formed by multiple proteins, their organization does not necessarily correspond to that of the individual proteins that make up the capsid shell. Even when structure is well preserved and a high degree of contrast can be achieved, the minimal size of an object that can be distinguished by classical electron microscopy, its resolution, is limited to 50 to 75 Å. Th is resolution is far too poor to permit molecular interpretation: for example, the diameter of an α-helix in a protein is on the or der of 10 Å. Cryo-electron microscopy (cryo-EM), in which samples are rapidly frozen and examined at very low temperatures in a hydrated, vitrified (noncrystalline, glass-like) state, preserves native structure. Because samples are not stained, this technique allows direct visualization of the contrast inherent to the virus particle, and it also enables higher resolution.

The symmetry of most virus particles facilitates analysis of individual images obtained by cryo-EM for reconstruction of two- or three-dimensional structure (Box 4.1). This approach can be complemented by cryo-electron tomography, in which two-dimensional images are recorded as the vitrified sample is tilted at different angles to the electron beam and subsequently combined into a three-dimensional density map (Fig. 4.3). These methods have been greatly improved since their introduction, to near atomic-level resolution, and are now standard tools of structural biology. Their application has provided unprecedented views of virus particles not amenable to other methods of structural analysis, for example, alphaviruses (Fig. 4.23) and herpesviruses (Fig. 4.27).

The first descriptions of the molecular interactions that dictate the structure of virus particles were obtained by X-ray crystallography (Fig. 4.4) (see the interview with Dr. Michael Rossmann: http://bit.ly/Virology_Rossmann). A plant virus (tobacco mosaic virus) was the first to be crystallized, and the first high-resolution virus structure determined was that of tomato bushy stunt virus. Since this feat was accomplished in 1978, high-resolution structures of increasingly large animal viruses have been determined, placing our understanding of the principles of capsid architecture on a firm foundation.