Principal Techniques for the Study of Virus Particle and Genome Structure

The study of a plant virus usually begins with investigation of two aspects, the structure of the virus particle and the organization of the viral genome. The most important methods used in such studies are outlined briefly in this chapter.

1 STRUCTURE OF VIRUS PARTICLES

The two major techniques for studying virus structure are X-ray crystallography and electron microscopy using various kinds of specimen preparation.

1.1 X-RAY CRYSTALLOGRAPHIC ANALYSIS

When X-rays pass through a crystal, the rays are scattered in a regular manner. The scattered radiation can be recorded photographically. What is recorded is, however, not a picture of the virus particle, but a very abstract-appearing collection of dots from which the structure of the virus particle is deduced by complex mathematical analysis. Inducing virus particles to form crystals suitable for X-ray crystallography is more of an art than a science, and generally requires many trials of salt and alcohol solutions and other precipitating conditions to obtain crystals of sufficient size and stability. Isometric particles will form true crystals. Rod-shaped, rigid virus particles often will form liquid crystals in which the rods are regularly arrayed in two dimensions. X-ray analysis can be applied to such crystals, but not to rod-shaped viruses with flexuous particles or to large virus particles with lipoprotein envelopes.

Where they can be applied, X-ray techniques provide the most powerful means of obtaining information about virus structure. Over the past 10 years, significant advances have allowed the application of X-ray crystallographic analysis to more viruses and at higher resolutions. With the definition of structures at atomic level, it has been possible to define interactions between the viral genome and the protecting protein coat, and to establish the positions of water molecules and divalent cations in the structure.

In summary, the major technical advances responsible for this progress have been (1) high-intensity, coherent X-ray sources that allow data to be recovered in a short time from delicate crystals; (2) an increase in the speed and capacity of computers, together with a reduced cost of computing; (3) noncrystallographic symmetry averaging, a process involving successive approximations that remove noise and enhance detail in the density map; and (4) the development of computer graphics, replacing the laborious manual model building that was required previously to refine structures.

There is a significant limitation for the study of small isometric viruses that can be crystallized. Such viruses crystallize because of regular symmetries in the protein shell. However, most of the nucleic acid inside the virus is not arranged in a regular manner with respect to these symmetries. Thus, very little information can usually be obtained about the conformation of the genome within the virus. Such information can be obtained for the rigid rod-shaped viruses, in which the RNA is arranged in a regular helix within a cylinder of protein.

1.2 ELECTRON MICROSCOPY

Development of images using electron microscopy depends on differences in electron scattering in different parts of the specimen. Virus particles themselves have very little contrast with respect to the scattering of electrons, compared with the carbon film on which they are usually mounted. For this reason, various specimen-preparation techniques have been used to enhance contrast. In early work, shadowing of the specimen at an angle with a vaporized heavy metal, such as gold, was employed. This procedure, however, obscured much detail. It was subsequently shown that various osmium, lead and uranyl compounds, and phosphotungstic acid (under certain conditions) react chemically with, and are bound to the virus. This procedure was called positive staining, but it was found to cause alteration in or disintegration of the virus structure. Today negative staining is the most widely used procedure for visualization of viruses in the electron microscope.

1.2.1 Negative Staining

Negative staining uses potassium phosphotungstate at pH 7.0, or uranyl acetate or formate near pH 5.0. The electron-dense material does not react chemically with the virus, but penetrates available spaces on the surface and within the virus particle. The virus structure stands out against the electron-dense background (Fig. 5.2). However, even in the best electron micrographs, fine details of structure tend to be obscured, first, by noise due to minor irregularities in the virus particle image and in the stain, and second, by the fact that contrast due to the stain is often developed on both sides of the virus particle to a varying extent. Thus electron micrographs of very high quality are essential in order to distinguish particles of small isometric viruses belonging to different virus groups. More detailed structural information may be obtained from a number of images of single negatively stained particles, by processing the image in one of several ways (e.g., Fig. 5.9).

1.2.2 Thin Sections

The diameter of small isometric viruses (20–30 nm) is much less than the thickness of a typical thin section (≈ 40–100 nm) used to study tissues by electron microscopy. Thus, no detailed structural information is revealed. However, some aspects of the structure of the larger viruses with lipoprotein envelopes can be studied using thin sections of infected cells (Fig. 5.16) or of a pellet containing the virus.

1.2.3 Cryoelectron Microscopy

Cryoelectron microscopy is a recently developed technique. The specimen is frozen extremely rapidly in an aqueous medium. The virus is suspended in a very thin film of liquid stretching across holes in a carbon grid, which is plunged into liquid ethane that contains some ethane ice (183 °C). The freezing is so rapid that water molecules do not have time to form micro ice crystals; thus, the specimen is frozen in vitreous ice with no damage caused by crystallization of water. The method is being usefully applied to the study of virus structures or substructures that may be altered by other specimen-preparation techniques.

2 THE STRUCTURE OF VIRAL GENOMES

2.1 CLASSICAL PROCEDURES

The nature of a viral nucleic acid, whether it is DNA or RNA, and whether it is single-stranded or double-stranded, circular or linear, can be established by various standard physical, chemical and enzymatic methods. Chemical and enzymatic procedures allow known special structures at the 5'- or 3'-end of linear genomic nucleic acid to be identified. Electrophoresis of purified nucleic acid from virus particles usually will give good estimates of the RNA or DNA molecular weight and the number of different size classes of genomic nucleic acid for those viruses with split genomes. The application of gene-manipulating technology is also rapidly increasing our understanding of the structure of viral genomes and how they replicate.

2.2 GENE-MANIPULATION TECHNOLOGY

2.2.1 Importance

There are two situations in which viral genome sequence information is of great use. Each of these situations has theoretical and practical aspects.

The Virus in the Plant

Theoretical A knowledge of the viral genes and the products they code for is beginning to lead to an understanding of how viruses cause disease.

Practical The ability to identify and isolate particular viral genes and integrate them into the host-plant genome is providing novel methods for understanding virus gene function and, in some instances, for the control of virus diseases.

The Virus in Relation to Other Viruses

Theoretical A knowledge of the nucleotide sequences of many viral genomes is of very great assistance in virus classification. The nucleotide sequences are revealing unexpected relationships between viruses, and this information is beginning to give us an understanding of how viruses might have originated and how they evolved. Computer- aided comparison of a viral nucleotide sequence with those of other viruses, and sequences encoding cellular proteins can sometimes indicate possible viral protein functions.

Practical It is essential to be...