Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 93

The use of centrifugal force to separate particles from solution according to size, shape, or density has been a staple of virology. The instrument used for such separations is called a centrifuge, which can range from small tabletop devices that accommodate small tubes to large floor models with greater capacity and to ultracentrifuges that can achieve revolutions per minute in excess of 70,000. The ultracentrifuge was invented by Theodor 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.