Two lines of evidence indicate that synthesis of viral DNA occurs in the cytoplasm of duck embryo fibroblasts infected with avian sarcoma virus: (i) viral DNA is detected first in the cytoplasmic fraction of infected cells and subsequently in the nuclear fraction; and (ii) viral DNA is synthesized at a normal irate in cells infected after enucleation with cytochalasin B. The presence of viral DNA in the. cytoplasmic fraction is not a consequence of leakage of newly synthesized viral DNA from the nucleus, since it remains in nuclear fractions late after infection when integration of viral DNA into the host genome is inhibited by ethidium bromide.After infection of susceptible cells by RNA tumor viruses, RNA of the input virus is transcribed into viral DNA, which is subsequently integrated into the host cell genome as provirus (1). The biochemistry of viral DNA synthesis, integration, and transcription is largely unknown. Studies with inhibitors-(2, 3) and direct measurement with molecular hybridization techniques (4,5) indicate that viral DNA synthesis occurs early after infection, but the intracellular site of its synthesis has not been unambiguously established (6)(7)(8).The present study was undertaken to establish the intracellular site of viral DNA synthesis by direct measurement in cytoplasmic extracts of infected cells and in infected enucleated cells. We find that early after infection viral DNA can be detected exclusively in the cytoplasmic fraction of the cell and that it later appears in the nucleus, where it eventually becomes covalently linked to the host genome (4). sonicator. Infection of intact or enucleated cells was carried out as described in the legends. Virus titers were determined by focus formation on chicken cell monolayers; titers were 2 to 10 X 106 focus-forming units (FFU)/ml for medium harvested from virus-producing cells and 1 to 5 X 108 FFU1 ml for concentrated virus. The efficiency of plating B77 on duck cells is about 40% relative to chicken cells.Cell Fractionatin. Cells were scraped into medium 199, collected by centrifugation, washed twice, and allowed to swell for 5 min at 00 in a hypotonic buffer containing 10 mM NaCl, 1 mM Tris HCl (pH 7.4), 0.15 mM MgC2. Deoxycholate and NP-40 were added to give a final concentration of 1.0% and 0.5%. (w/v), respectively, and the cells were broken by five strokes in a glass Dounce homogenizer. The disrupted cells were centrifuged at 800 X g for 10 min and the supernatant (cytoplasm) was separated from pelleted nuclei. Alternatively, the cells were swollen for 5 Mini NaClTris-HCl-MgCl2 buffer containing 0.5% NP40, broken, and centrifuged as above. The two methods gave similar results.Cell Enucleation. -Primary cultures of duck embryo fibroblasts were trypsinized and seeded at a density of about 2 X 105/cm2 on plastic disks cut from 75-cm2 T-flasks (Falcon). Before use, the disks were gently rubbed with fine sandpaper and exposed to concentrated sulfuric acid for 10 miil to augment cell adhesion; they were then washed with distilled wa...
Labeled, virus-specific DNA synthesized in vitro by the virion-associated polymerase of avian sarcoma virus (ASV) was used to measure virus-specific sequences in cell DNA in three ways: (i) by determining the effect of cell DNA upon the reassociation rate of double-stranded polymerase products; (ii) by measuring the kinetics of annealing of single-stranded polymerase product (cDNA) to cell DNA; or (iii) by measuring the amount of cDNA which anneals to a large excess of cell DNA. With these three assays and modifications of them, we show that fewer than five copies of ASV-specific DNA sequences are present per diploid cell in uninfected chicken embryos; that a two-to several-fold increase in copy number of viral DNA follows infection by ASV; that infection introduces to the cell viral sequences not present before infection; and that DNAs from uninfected Pekin duck and Japanese quail embryos show no homology with DNA synthesized by the ASV polymerase. Some of these results differ from data in a previous report from this laboratory (
We have attempted to distinguish integrated and unintegrated forms of avian sarcoma virus-specific DNA in cells by sedimentation through an alkaline sucrose gradient in a slowly reorienting zonal rotor. Results obtained with this procedure are similar to those obtained by the more convenient analysis of networks of high-molecular-weight cell DNA. Most, if not all, viral DNA appears completely integrated into the host cell genome in an avian sarcoma virustransformed mammalian cell and in normal chicken cells (in which viral DNA is genetically transmitted). Fully transformed duck cells and duck embryo fibroblasts infected for 20 to 72 h contain both integrated and unintegrated viral DNA; up to one copy per cell is integrated within 20 h after infection, and four to eight copies are integrated in fully transformed cells. The amount of unintegrated DNA varies but may comprise over 75% of the viral DNA in acutely infected cells and from 20 to 70% ofthe viral DNA in fully transformed cells. The unintegrated DNA in either case consists principally of duplexes with "minus" strands the length of a subunit of the viral genome (2.5 x 106 to 3 x 106 daltons) and relatively short "plus" strands (0.5 x 106 to 1.0 x 106 daltons).
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