In vitro reassembly of shell-like particles from disrupted polyoma virus.
When purified polyoma virus is exposed to 0.01 M dithiothreitol in the presence of 0.2 M Na(2)CO(3)-NaHCO(3) (pH 10.6) at 0-4 degrees C, the capsids are rapidly disrupted to protein subunits of capsomere size, as judged by density gradient centrifugation, sedimentation equilibrium centrifugation, and electron microscopy. Hemagglutination activity and infectivity of disrupted virus are reduced to below detectable amounts. Removal of the disruption reagents by dialysis at 4 degrees C against 0.05 M Tris-0.14 M NaCl-1 mM EDTA and 0.1 mM 2-mercaptoethanol (pH 8.0) results in a time-dependent reappearance of up to 17% of the starting hemagglutination titer, under optimum conditions of ionic strength, pH, temperature, and virus protein concentration. The recovered hemagglutination activity is found in glycerol gradients associated with a 100S DNA-protein complex consisting mostly of linear aggregates of capsomeres. When the linear complex is treated with pancreatic DNase, the complex is converted into spherical particles, of approximately virus size, that sediment at 140 S (with aggregates at 180 S), as well as on the cushion of half-saturated CsCl at the bottom of the gradients. All reassembled particles are not infectious and have markedly reduced DNA to protein ratios.
D000818:Animals; D001345:Autoradiography; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral / Q000032:analysis; D003851:Deoxyribonucleases / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D011120:Polyomavirus / Q000032:analysis* / Q000187:drug effects / Q000254:growth & development; D013696:Temperature; D014316:Tritium; D014764:Viral Proteins / Q000032:analysis*; D014774:Virulence
D002247:Carbon Isotopes; D004279:DNA, Viral; D014764:Viral Proteins; D014316:Tritium; D003851:Deoxyribonucleases; D004229:Dithiothreitol