Virus purification by CsCl gradient centrifugation.

PSVs, FCV, EV70, RV, and CVB3 grown in each permissible cell line were purified by CsCl gradient centrifugation. Infected cell cultures harvested at 72 h postinoculation were freeze-thawed three times, and cell debris was spun down at 2,469 × g for 10 min at 4°C. A total of 500 ml of virus-containing supernatants was concentrated by centrifugation at 245,853 × g for 20 h at 4°C using an SW40 rotor (Beckman). The viruses in the pellets were resuspended in TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 100 mM EDTA [pH 7.5]), and then the suspension was layered over 29 to 41% preformed discontinuous CsCl gradients. After centrifugation at 245,853 × g for 20 h at 4°C using an SW40 rotor (Beckman), the banded virus was collected by puncturing the side of the tube with a needle, diluted in distilled water, and further purified by ultracentrifugation at 245,853 × g for 20 h at 4°C in an SW40 rotor (Beckman). Purified viruses were dialyzed into 0.1 M sodium bicarbonate buffer (pH 8.3) for fluorescence labeling or in TNE buffer for a radioactivity assay overnight and then stored in aliquots at −80°C. Analysis of SDS-PAGE-separated, [³⁵S]methionine-cysteine-labeled viral particles by radiography, Western blotting, or Coomassie blue staining showed that the label was exclusively coupled to the viral capsid protein (data not shown). Plaque assays showed no loss in infectivity with each labeled virus in each permissible cell line in comparison with unlabeled virus (data not shown). In a CsCl gradient purification, the supernatant of an RV strain Wa-infected culture generated two distinct visible bands (29). Each banded virus was analyzed by the above-listed methods, and virus purified from the fraction with infectious triple layered particles by CsCl gradient centrifugation was used for a radioactivity assay (data not shown).