Principles of Virology, Volume 1. Jane Flint
2.11). Because mRNAs undergoing translation can be associated with different numbers of ribosomes, they can be separated on a sucrose gradient. A more modern use of the polysome profile is to extract the RNA from each fraction and determine which mRNAs are being actively translated.
Another method for purifying viruses is by isopycnic centrifugation, which separates particles solely on the basis of their density. A virus preparation is mixed with a compound (e.g., cesium chloride) that forms a density gradient during centrifugation. Virus particles move down the tube until they reach the point at which their density is the same as the gradient medium. Structural studies of virus particles often require highly purified preparations which can be made by differential or isopycnic centrifugation.
Figure 2.11 Polysome analysis. To study the association of mRNAs with ribosomes, cell lysates are prepared and separated by centrifugation through sucrose gradients. Fractions are collected and their optical density measured to locate mRNAs bound to one or more ribosomes. The graph shows the optical density of fractions from the top (left) to the bottom (right) of the gradient. The slower-moving materials at the top of the gradient are ribosomal subunits, while mRNAs associated with one or more ribosomes move faster in the sucrose gradient.
Measurement of Viral Enzyme Activity
Some animal virus particles contain nucleic acid polymerases, which can be detected by mixing permeabilized particles with precursors and measuring their incorporation into nucleic acid. This type of assay is used most frequently for retroviruses, many of which neither transform cells nor form plaques. The reverse transcriptase incorporated into the virus particle is assayed by mixing cell culture supernatants with a mild detergent (to permeabilize the viral envelope), an RNA template and primer, and a radioactive nucleoside triphosphate. If reverse transcriptase is present, a radioactive product will be produced by priming on the template. This product can be detected by precipitation or bound to a filter and quantified. Because enzymatic activity is proportional to particle number, this assay allows rapid tracking of virus production in the course of an infection. Many of these assays have been modified to permit the use of safer, nonradioactive substrates. For example, when nucleoside triphosphates conjugated to biotin are used, the product can be detected with streptavidin (which binds biotin) conjugated to a fluorochrome. Alternatively, the reaction products may be quantified by quantitative real-time PCR (see “Detection of Viral Nucleic Acids” below).
Serological Methods
The specificity of the antibody-antigen reaction has been used to design a variety of assays for viral proteins and antiviral antibodies. These techniques, such as immunostaining, immunoprecipitation, immunoblotting, and the enzyme-linked immunosorbent assay, are by no means limited to virology: all these approaches have been used extensively to study the structures and functions of cellular proteins.
Virus neutralization. When a virus preparation is inoculated into an animal, an array of antibodies is produced. These antibodies can bind to virus particles, but not all of them can block infectivity (neutralize), as discussed in Volume II, Chapter 4. Virus neutralization assays are usually conducted by mixing dilutions of antibodies with virus, incubating them, and assaying for remaining infectivity in cultured cells, eggs, or animals. The end point is defined as the highest dilution of antibody that inhibits the development of cytopathic effect in cells or virus reproduction in eggs or animals.
Some neutralizing antibodies define type-specific antigens on the virus particle. For example, the three serotypes of poliovirus are distinguished on the basis of neutralization tests: type 1 poliovirus is neutralized by antibodies to type 1 virus but not by antibodies to type 2 or type 3 poliovirus. The results of neutralization tests were once used for virus classification, a process now accomplished largely by comparing viral genome sequences. Nevertheless, the detection of antiviral antibodies in animal sera is still extremely important for identifying infected hosts. These antibodies may also be used to map the three-dimensional structure of neutralization antigenic sites on the virus particle (Box 2.7).
DISCUSSION
Neutralization antigenic sites
Antigenic sites defined by antibodies. (A) Locations of neutralization antigenic sites on the capsid of poliovirus type 1. Amino acids that change in viral mutants selected for resistance to neutralization by monoclonal antibodies are shown in white on a model of the viral capsid. These amino acids are in VP1 (blue), VP2 (green), and VP3 (red) on the surface of the virus particle. Figure courtesy of Jason Roberts, Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia. (B) Conformational and linear epitopes bound to antibody molecules. Linear epitopes are made of consecutive amino acids, while conformational epitopes are made of amino acids from different parts of the protein.
Knowledge of the antigenic structure of a virus is useful in understanding the immune response to these agents and in designing new vaccination strategies. The use of monoclonal antibodies (antibodies of a single specificity made by a clone of antibody-producing cells) in neutralization assays permits mapping of antigenic sites on a virus particle or of the amino acid sequences that are recognized by neutralizing antibodies.
Each monoclonal antibody binds specifically to 8 to 12 residues that fit into the antibody-combining site. These amino acids are either next to one another either in primary sequence (linear epitope) or in the folded structure of the native protein (nonlinear or conformational epitope). In contrast, polyclonal antibodies comprise the repertoire produced in an animal against the many epitopes of an antigen. Antigenic sites may be identified by cross-linking a monoclonal antibody to the virus and determining which protein is the target of that antibody. Epitope mapping may also be performed by assessing the abilities of monoclonal antibodies to bind synthetic peptides representing viral protein sequences. When the monoclonal antibody recognizes a linear epitope, it may react with the protein in immunoblot analysis, facilitating direct identification of the viral protein harboring the antigenic site.
An elegant understanding of antigenic structures has come from the isolation and study of variant viruses that are resistant to neutralization with specific monoclonal antibodies (called monoclonal antibody-resistant variants). By identifying the amino acid change(s) responsible for this phenotype, the antibody-binding site can be located and, together with three-dimensional structural data, can provide detailed information on the nature of antigenic sites that are recognized by neutralizing antibodies (see the figure).
Hemagglutination inhibition. Antibodies against viral proteins with hemagglutination activity can block the ability of virus to bind red blood cells. In this assay, dilutions of antibodies are incubated with virus, and erythrocytes are added as outlined above. After incubation, the titer is read as the highest dilution of antibody that inhibits hemagglutination. This test is sensitive, simple, inexpensive, and rapid, and can be used to detect antibodies to viral hemagglutinin in animal and human sera. For example, hemagglutination inhibition assays were used to identify individuals who had been infected with the newly discovered avian influenza A (H7N9) virus in China during the 2013 outbreak.
Visualization of proteins. Antibodies can be used to visualize viral or cellular proteins in infected cells or tissues. In direct immunostaining, an antibody that recognizes a viral protein is coupled directly to