Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.