Treatment of polyoma virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP4-7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of VP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP,, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP-7 were found to be predominantly associated with the 5S capsomere subunit.

Analysis of polyoma virions by X-ray fluorometry demonstrated that calcium (Ca2+) was associated with the purified virion. Treatment of purified virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, and the reducing agent dithiothreitol caused the virions to dissociate. Electron microscopy revealed that the virions were dissociated to the capsomere level. Incubation of polyoma virions with 150 mM NaCl, 10 mM EGTA, and 3 mM dithiothreitol was optimum for the dissociation reaction. The pH for the dissociation reaction ranged from 7.5 to 10.5. Cesium chloride density gradient centrifugation indicated that both EGTA and dithiothreitol were necessary for dissociation to occur; neither reagent alone dissociated the virus. The major protein product of the dissociated viral particles sedimented at 12S. Relationships between these experiments and the alkaline carbonate-bicarbonate dissociation of polyoma are discussed.

The plaque-assay technique was used as a tool to determine the optimal conditions for adsorption of polyoma virions to host cells. Using these optimal conditions of adsorption, an electron microscopy study of the early events of infection was performed. By electron microscopy and autoradiography, it was demonstrated that both the viral coat proteins and DNA arrive simultaneously in the nucleus as early as 15 min postinfection. When horseradish peroxidaselabeled virions, pseudovirions, and capsids were used to infect cells, only the particles with nucleic acid or a factor(s) associated with the nucleic acid, i.e., histones, appeared to enter the nucleus. Moreover, when virions were used to infect either permissive or nonpermissive cells, identical early events of viral infection, i.e., adsorption, penetration, and nuclear transport, were observed, suggesting that these early events of infection are a property of the virion and not the host cell.

Segregation of sister chromatids in embryonic mouse cells in primary tissue culture is not random. In mitosis those chromatids replicated on a DNA template synthesized during the preceding division cycle are separated from those constructed on a template synthesized two division cycles previously. Segregation in cells of the Chinese hamster follows a similar, but less pronounced, pattern.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP, was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP, was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP, appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP, species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP, species and three of the capsid VP, species were found in capsomere preparations enriched for hexon subunits. Two VP, species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP, species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP, species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.