INTRACELLULAR STATE OF MAREKS-DISEASE VIRUS-DNA IN 2 TUMOUR-DERIVED CHICKEN-CELL LINES

The intracellular state of Marek's disease virus (MDV) DNA was investigated in two permanent chicken cell lines: HPRS-I and MSB-I. The HPRS-I line was established from an ovarian lymphoma of a chicken with Marek's disease and is a virus-non-productive line, while the MSB-I line originates from another animal with a Marek's disease splenic lymphoma and is a low producer line. By repeated isopycnic centrifugation in CsCl, MDV DNA in the HPRS-I line showed properties of integrated DNA, whereas in cells of the productive MSB-I line both integrated and free virus DNA appeared to be present. Under denaturing conditions (0.1 M-NaOH) the virus DNA remained associated with the cellular DNA as revealed by equilibrium centrifugation in CsCl and hybridization of the DNA in each single fraction with 32P-labelled complementary RNA transcribed from the DNA of the GA strain of MDV. Shearing of HPRS-I DNA to a mol. wt. of about 8 x 106 released only part of the virus DNA to the density of free virus DNA, while a large proportion of MDV DNA could still be localized at the density of cellular DNA. Sedimentation velocity experiments with HPRS-I and MSB-I DNA originally fractionated on CsCl gradients revealed integrated virus DNA sequences in both cell lines and an additional peak of virus DNA at the position of free linear MDV DNA in the MSB-I line. No fast-sedimenting virus DNA molecules with properties of covalently closed circular structures could be detected. Further evidence for the presence of integrated virus DNA sequences in MDV-transformed cells was provided by the Hirt (1967) precipitation procedure.