RD-114 virus: Analysis of viral gene sequences in feline and human cells by DNA-DNA reassociation kinetics and RNA-DNA hybridization

Abstract

RD-114 virus is a C-type virus found growing in a human rhabdomyosarcoma cell line (RD) after inoculation into a fetal cat. To investigate the host species of origin of RD-114 virus and its relationship to other oncornaviruses, we performed molecular hybridization and reassociation kinetic experiments using three radioactive probes—the double stranded (ds) and single stranded (ss) DNA products of the viral RNA-directed DNA polymerase and the viral 60–70 S [ 3H ]RNA genome. DNA was synthesized by the endogenous DNA polymerase reaction of purified RD-114 virions disrupted with detergent and dsDNA was isolated by hydroxyapatite chromatography. The most efficiently transcribed dsDNA segment represented approximately 50% or more of dsDNA transcripts and had a sequence complexity of about 0.8 × 10 6 daltons. The kinetics of reassociation of this rapidly reassociating dsDNA segment in the presence of cellular DNA was used to detect viral gene sequences in human and feline cells. The RD-114 cell line which chronically produces RD-114 virus contains 20–30 copies of this DNA sequence while normal feline tissues contain 100–200 copies. Less than one copy of DNA sequences homologous to this RD-114 viral dsDNA segment were detected in uninfected human, hamster, and rat cells. RD-114 viral [ 3H]ssDNA did not hybridize significantly with RNA of several strains of feline and murine leukemia and sarcoma viruses. RD-114 viral 60–70S [ 3H]RNA hybridized to the same extent and with similar kinetics to RD-114 cell and feline tissue DNA indicating that similar numbers of copies of most, if not all, of RD-114 viral genome, are present in normal feline cells as well as in RD-114 virus-producing human cells. No hybridization was detected between RD-114 viral 60–70S RNA and the DNA of the parental human RD cell line, or the DNA of uninfected human, hamster or rat cells. These measurements indicate that RD-114 virus incorporates less than 10% human cell DNA sequences even after replicating in human cells for many generations. We conclude that the major portion of the RD-114 virus genome is of feline derivation, that it shares base sequences with a repetitive class of normal feline cell DNA, and that it is genetically distinct from known isolates of feline leukemia-sarcoma viruses. These properties suggest that RD-114 virus is an endogenous virus of normal feline cells.