Covalently closed circular DNA of avian sarcoma virus: Purification from nuclei of infected quail tumor cells and measurement by electron microscopy and gel electrophoresis

Abstract

Avian sarcoma virus-specific DNA is synthesized three to fourfold more efficiently after infection of a Japanese quail tumor cell line (QT-6, derived from a fibrosarcoma induced with methylcholanthrene) than after infection of duck or quail embryo fibroblasts; 10 to 30 copies of viral DNA per cell are generally observed in the first 24 hours after infection at multiplicities of one to five focus-forming units per cell. During the first five hours after infection viral DNA accumulates only in the cytoplasm of QT-6 cells; thereafter viral DNA is also observed in the nucleus. Covalently closed circular forms of viral DNA (form I) accumulate exclusively in the nucleus, and they persist for at least 600 hours after infection; within 24 to 48 hours after infection, form I DNA constitutes as much as 50% of the nuclear species of viral DNA and 20 to 25% of viral DNA in whole cells. We have purified viral form I DNA about 10 5-fold from acutely-infected QT-6 cells by cell fractionation, sodium dodecyl sulfate/sodium chloride precipitation of cellular DNA, equilibrium density centrifugation in cesium chloride gradients containing propidium diiodide, rate zonal sedimentation in sucrose gradients, and chromatographic selection of molecules renaturing with zero-order kinetics. The length of viral form I DNA has been measured by direct observation in the electron microscope, revealing species of 6.6 × 10 6, 5.6 × 10 6 and 2 to 4 × l0 6 daltons. These species presumably correspond to DNA transcribed from RNA subunits of avian sarcoma virus (3.3 × 10 6 daltons); DNA transcribed from RNA subunits of transformation-defective deletion mutants of the virus (2.8 × 10 6 daltons) shown to be present in the infecting stock; and newly identified, incomplete transcripts of these RNA subunits. The size of these species and their viral specificity were confirmed by molecular hybridization after electrophoresis in agarose gels. Viral form I DNA of 5 to 7 × 10 6 daltons is infectious in chick embryo cells, resulting in production of avian sarcoma virus and transformation-defective virus, whereas viral form I DNA of 2 to 4 × 10 6 daltons is not infectious in similar tests and is presumably defective.