Synthesis of Viral DNA in the Cytoplasm of Duck Embryo Fibroblasts and in Enucleated Cells after Infection by Avian Sarcoma Virus

Abstract

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...

Infectious viral DNA of murine leukemia virus.

“…Following infection by RNA tumor viruses, various forms of unintegrated viral DNA appear and can be detected by molecular hybridization (1)(2)(3). Some of these forms, including the closed-circular, supercoiled form, are probable precursors to the integrated viral genome.…”

Molecular Relationship of the DNA and RNA of Intracytoplasmic A Particles to Mouse and Mammary Tumour Virus Genomes

“…Two variations of the procedure were used. In the first (Varmus et al 1974), the DNA was dissolved in o'3 N-NaOH, o'oo5 M-EDTA and heated in a boiling water bath for 2o min. It was neutralized with acetic acid, the DNA was precipitated out by ethanol and the size of the DNA fragments determined as described below.…”

Isolation and Characterization of Host Range Mutants of Avian Sarcoma Virus

“…Duck embryo fibroblasts (DEF) seem useful for studying the genetics of avian sarcoma virus (ASV), since they contain no detectable endogenous viral genome that can hybridize with avian leukosis virus (Varmus et al, 1973(Varmus et al, , 1974Tereba et al, 1975;Stavnezer et al, 1976). However, one problem found when using DEF is that the efficiency of infection by ASV is low (Duff & Vogt, 1969;Altaner & Temin, 1970;Shimakage et at., 1979).…”