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

Abstract

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.3Earlier 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.4For 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-5-H3 (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 t...

Maturation of Papillomavirus Capsids

“…Nucleasetreated HPV16 and HPV18 L1/L2 vector stocks contained substantial amounts of encapsidated cellular DNA (Fig. 5), reminiscent of early reports of adventitious uptake of cellular DNA fragments by polyomaviruses (24,33) and more recent reports of encapsidation of cellular DNA by HPV33 capsids (12). The presence of encapsidated cellular DNA fragments implies that, prior to cell lysis, papillomavirus procapsids can exist in a partially assembled state around loops of cellular DNA.…”

Induction and Bypass of p53 during Productive Infection by Polyomavirus

“…Two other mechanisms of induction are possible but less likely. One is that the introduction of linear fragments of cellular DNA via pseudovirions (42) would mimic the generation of double-strand breaks by ionizing radiation and thereby induce a p53 response. This seems unlikely to be the only mechanism, since the RB1 mutant is impaired in its ability to induce the response.…”

Degradation of Pseudoviral DNA after Infection of Mouse Cells with Polyoma Pseudovirions

“…They have been found in baby mouse kidney eells (1,5,6), primary mouse embryo cells (2-4, 6, 7), mouse 3T3D cells (6) and mousehamster somatic cell hybrids (8) infected with polyoma virus. The pseudoviral DNA molecules are linear and doublestranded and have a molecular Weight of about 3 X 106 (1,3,5).…”

“…Polyoma pseudovirions contain fragments of host cell DNA encapsidated within polyoma protein coats (1)(2)(3)(4)(5). They have been found in baby mouse kidney eells (1,5,6), primary mouse embryo cells (2-4, 6, 7), mouse 3T3D cells (6) and mousehamster somatic cell hybrids (8) infected with polyoma virus.…”

“…Polyoma pseudovirions have been suggested as potential agents for mammalian gene transfer or transduction (1,(9)(10)(11)(12). However, attempts to introduce into deficient cells the genes for thymidine kinase and inosinic pyrophosphorylase using polyoma pseudovirions have been unsuccessful (11).…”