Isolation of structural proteins of the virus

A large number of techniques are available for fractionation of biological molecules and subcel-lular particles according to their size, density, or charge. Buoyant density differences are useful in fractionating enveloped viruses. Each subcellular particle has differences in buoyant density in aqueous solution. Those with large membrane components are "lighter" than those composed of only proteins and nucleic acids.

Virus particles also can be separated from cellular components of different density. This is accomplished by generating an equilibrium density gradient of sucrose or other material in an ultracentrifugal field. Virus particles will "band" or "float" at a specific location within the gradient corresponding to their equilibrium buoyant density (1.18 g/cm3 in the example shown in Fig. 11.1). This position represents a balance of forces on the particle: the buoyant force trying to cause the particle to float and the centrifugal force working to cause the particle to sediment lower in the gradient.

Size fractionation is widely used, especially for nonenveloped viruses. For subcellular particles, organelles, and virions, differential sedimentation under a centrifugal field (rate zonal centrifugation) allows rapid fractionation and purification. In essence, one takes advantage of the difference in size of these components in the centrifugal field where the largest (the ones with the greatest sedimentation coefficient) will sediment most rapidly or under the least force. The practical aspects of such differential centrifugation can be complex. The basic approach is readily seen in Fig. 11.2. Since most viruses are smaller than mitochondria and larger than

Fig. 11.1 Equilibrium density gradient centrifugation of virus-infected cell components to isolate virus particles. A preformed sucrose density gradient is layered with a solution of infected cell material and subjected to centrifugation at high g force at 4°C for several days. Virus particles sediment downward until they reach a layer with a density equivalent to their own. At this density, the virus particles will "float" and careful handling of the gradient in a clear plastic tube will reveal a turbid band of virions that can be removed. In the figure shown, the virus was collected by careful dropwise fractionation of the gradient through a hole in the tube bottom into small tubes. The presence of virus in the appropriate fractions could be confirmed by plaque assay.

Sucrose density

  1. 04 g/cm3
  2. 28 g/cm3

Sample m

Virus will "float" at its buoyant density ooooo

Centrifuge until equilibrium is reached

600 X g

10 min

Filtered homogenate

Nuclei in pellet

15,000 X g 15 min

Mitochondria, chloroplasts, lysosomes and peroxisomes

100,000 X g 15 min

Fig. 11.2 Differential centrifugation to purify virions. Infected cells are homogenized and then subjected to varying steps of centrifugation at increasing g forces. At low speeds, large cellular components pellet and can be removed. At the proper speed, viral particles sediment to the bottom of the tube.


Plasma membrane, microsomal fraction

  • Supernantant
  • 300,000 X g <-■-—

30-60 min

Virus in pellet (also ribosomes small orangelles)

Virus will remain in suspension during low "g" spins ribosomes, further fractionation could be obtained by taking the 100,000g supernatant material and carrying out further differential centrifugation or more careful size fractionation.

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