Biological activity of cloned rat endogenous C-type virus DNA transferred by microinjection

The biological activity of a molecularly cloned DNA of a rat endogenous C-type leukaemia helper virus, RHHV, was assessed by intranuclear microinjection into normal rat kidney cells (NRK153). Release of rat C-type leukaemia helper viruses by the microinjected cells was examined by superinfection of Kirsten-transformed non-producer cells (K-NRK). Immediate release of helper leukaemia viruses at a very low level was observed only in the NRK153 m3 . 5/cir cells microinjected with the supercoiled form of RHHV DNA in toto, suggesting that the circular form of the virus DNA might have expedited the replication and expression of virus particles. Genome rescue experiments were also performed by co-cultivating the microinjected NRK153 m cells carrying various linear RHHV DNAs, in toto or of subgenomic sizes, with K-NRK cells. The results indicated that both the total and the 5.8 to 6.2 kb DNA fragment proximal to the 5' terminus of the cloned RHHV 8.8 kb DNA were able to rescue successfully a transforming replication-competent pseudotype virus. Subgenomic DNA fragments derived from the centre or the 3' end of the RHHV DNA were ineffective in the genome rescue experiments.