Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 210

As illustrated in the preceding sections, studies of purified virus particles by methods such as X-ray crystallography and cryo-EM can yield high-resolution descriptions of the interactions among particle components responsible for the assembly and sturdiness of these nanomachines. Such studies are typically performed under extreme conditions, for example, very low temperature, and the structures described are based on the averaging of very large numbers of particles. Consequently, these approaches provide no information about the dynamic or mechanical properties that underlie the functions of virus particles. Such information can be collected by various biophysical methods, including small-angle X-ray scattering and calorimetry. Atomic force microscopy, which permits both imaging of virus particles and measurement of mechanical properties, has been especially useful.

In this method, a very sharp tip attached to a cantilever scans the surface of a sample immobilized on a solid support (Fig. 4.30A). Deflections of the cantilever to or from the surface during scanning are detected by a laser beam via a position-sensitive detector. In this way, the topography of the surface of a sample such as a virus particle can be imaged, albeit at relatively low resolution (Fig. 4.30B). To assess mechanical properties, the tip is applied (nanoindentation) to a specific point on the surface, such as an axis of icosahedral symmetry, to deform the particle. Measurement of the force applied as a function of distance (degree of indentation) allows measurement of such parameters as elasticity and the force required to break the particle at that point. This approach has led to fascinating insights into how virus particles meet the seemingly paradoxical requirements for high stability to protect viral genomes and efficient disassembly during entry into a host cell.

Figure 4.30 Atomic force microscopy and its application to human adenovirus particles. (A) A schematic illustration of nanoindentation of virus particles. Virus particles are attached to a solid surface, such as a glass coverslip or mica, and the tip of an atomic force microscope (chosen to be smaller than the radius of the particles of interest) brought into contact. The tip is attached to a cantilever that moves to or from the surface of the particle as it is scanned. Deflections of a laser beam focused on the tip during scanning are recorded via a photodiode, allowing construction of an image of the particle surface. When the force applied via the tip is increased, the degree of deformation of the particle is measured as a function of force, as illustrated in the center. As the force increases, the virus particle eventually breaks, leading to a sudden drop in resistance (bottom). This force is often called the breaking force. Courtesy of G. Nemerow, The Scripps Research Institute. (B) Shown in the right two panels are atomic force microscopy images of a particle of human adenovirus type 5 with short fibers derived from type 35 before and after application of sufficient force to induce mechanical failure. The degree of resolution is low, but sufficient to determine the orientation of these icosahedral particles on the surface. To characterize mechanical failure in more detail, the responses of individual particles were scored on the basis of whether an edge, a facet, or a vertex was probed, revealing three distinct patterns (left three panels). This approach established that the vertices are the weakest points, exhibiting the lowest spring constant and breaking force. Adapted from Snijder J et al. 2013. J Virol 87:2756–2766.