Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells.
A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species.
D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D011120:Polyomavirus / Q000235:genetics* / Q000648:ultrastructure; D013936:Thymidine / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
D014764:Viral Proteins; D013936:Thymidine