CHARACTERIZATION OF CELL-LINES THAT INDUCIBLY EXPRESS THE ADENOASSOCIATED VIRUS REP PROTEINS

The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular t ransformation induced by various oncogenes. To study the rep gene's an tiproliferative effects, we have developed cell lines which express th e replication proteins under the control of an inducible mouse metallo thionein transcription promoter. The Rep78 protein produced in these c ell lines binds to the AAV terminal repeat sequences in vitro and supp orts AAV DNA replication and trans activation of the AAV p40 transcrip tion promoter in vivo. These cell lines are capable of assembling infe ctious viruses containing a mutant rep gene or a vector bearing a hete rologous gene. Growth rate and colony formation efficiency assays indi cated that rep gene expression substantially altered cellular prolifer ation, Long-term induction of the cell lines followed by removal of th e inducing agent suggested that constitutive expression of the Rep pro teins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicate d that the presence of the Rep proteins increased the population of ce lls in the S phase of the cell cycle. Thus the rep gene's antiprolifer ative effects may be realized by interference with cellular DNA replic ation.