Mouse cellular DNA enclosed in polyoma viral capsids (pseudovirions).

The DNA extracted from purified preparations of polyoma (Py) virus consists of three components, I, TI, and III, with sedimentation coefficients of 20, 16, and -14S. I and II are circular DNA molecules with a molecular weight of 3 X 106f and a buoyant density in CsCl of 1.709 gm cm-3.1, 2 Both I and II are infective.3 Earlier observations suggested that Py DNA III might be derived from mouse cellular DNA.4, 5 In this paper we will show that most of the DNA molecules in III are linear fragments of mouse cellular DNA enclosed in Py viral capsids. Viral particles that contain mouse DNA instead of the viral genome are not infective and are referred to as Py pseudovirions. Materials and Methods. -Wild-type virus was grown on confluent MK cell cultures4 6 and was harvested at various times after infection. The crude viral lysates were extensively treated with pancreatic DNase'8 and RNase'8 and then purified by differential and CsCl density gradient equilibrium centrifugation.6 If viral preparations were subjected to a second cycle of purification, the relative amounts of Py DNA 1, II, and III remained unchanged. '8 Plaque-forming titer was generally determined on MK cultures7 and in some comparative experiments on mouse embryo cultures.8 Hemagglutination titer was determined according to Eddy et al.9' 18 DNA was extracted from purified viral preparations with 0.6% SDS-0.01 M EDTA.4 For radioactive labeling of viral and cellular DNA deoxythymidine-methyl-Hi (TdR-H3, spec. act. 10-17 c/mM) and deoxythymidine-2-C14 (spec. act. 30 mc/mM) were used. '8 Radioactivity was measured on filter papers in a liquid-scintillation counter (Nuclear Chicago 6851). Unless otherwise indicated, the counting background was not subtracted. Sedimentation velocity analysis by band centrifugation and CsCl density gradient equilibrium centrifugation were performed according to methods used in earlier work. '0'" The buoyant densities for the DNA's were determined by using E. coli DNA as a standard (p = 1.710 gm cm-3). "2 The buoyant density of fractions (single drops) collected from CsCl density gradient of viral preparations was obtained from measurements of the refractive index at 250C. The value of 1.330 gm cm-' for Py virions (peak fraction) is the average from the measurement of 58 different viral preparations. DNA was examined by electron microscopy according to Kleinschmidt and Zahn. "3 Spherules of polystyrene latex (Dow Chemical) were added to the preparations and their shadow was used to calibrate the magnification. '4 The preparations were photographed at a magnification of 104. "Natural" mouse RNA for hybridization experiments was extracted with hot phenol"' 16 from uninfected MK cultures (2 days after plating) which had been labeled for 30 min with uridine-5H3 (5 pc/ml; spec. act. 4.4 c/mM) prior to the extraction. "Synthetic" mouse RNA was prepared in a cell-free system with DNA-dependent E. coli RNA polymerase (sed. const. 13S; E. coli MRE 600; RNase I-) according to Pettijohn and Kamiyal7 using highly purified DNA from MK cultures'8 as the primer. Under the conditions used, the attachment of the enzyme to the DNA was specific and enzymatic activity was optimal. '7 DNA and radioactive mouse RNA were hybridized in solution. The amount of DNA-RNA hybrids resistant to RNase was determined on nitrocellulose filters (Schleicher and Schuell, Bac-T-Flex). '6'19 Highly purified E. coli DNA was a gift of Dr. Bruce Alberts. Results. -(1) The behavior of Py virions and of pseudovirions in CsCl equilibrium density gradients: In CsCl density gradients Py viral hemagglutinin is present in two main bands (Fig. 1): The dense band contains infective Py virions ("virus band," p = 1.330 gm cm-3) while the light one consists mainly of viral capsids devoid