Svedberg in 1925, and it is the first initial of his last name that is used to describe the sedimentation coefficient of a particle as measured by centrifugation, e.g., the 16S ribosomal subunit.
Figure 2.10 Hemagglutination assay. (Top) Samples of different influenza viruses were diluted, and a portion of each dilution was mixed with a suspension of chicken red blood cells and added to the wells. After 30 min at 4°C, the wells were photographed. Sample A does not contain virus. Sample B causes hemagglutination until a dilution of 1:512 and therefore has a hemagglutination titer of 512. Elution of the virus from red blood cells at the 1:4 dilution is caused by neuraminidase in the virus particle. This enzyme cleaves N-acetylneuraminic acid from glycoprotein receptors and elutes bound viruses from red blood cells. (Bottom) Schematic illustration of hemagglutination of red blood cells by influenza virus. Top, Courtesy of C. Basler and P. Palese, Mount Sinai School of Medicine of the City University of New York.
It would not be wrong to state that every virology laboratory is in possession of at least one centrifuge and probably has access to more. The uses of the centrifuge in virology are manifold: from low-speed separation of virus particles from infected cell debris in cell culture medium to fractionation of infected cells to isolate nuclei, cytoplasm, or ribosomes, and to purification of virus particles.
Differential centrifugation is used to separate viruses, organelles, or subcellular structures from cells. Preformed gradients of sucrose are often used because particles that move with various velocities can be separated differentially in the increasing viscosity of the solution. One application of sucrose gradients is the purification of virus particles. Another is polysome profiling, an analysis of the mRNAs associated with ribosomes (Fig. 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
