DIRECTED INTEGRATION OF MINUTE VIRUS OF MICE DNA INTO EPISOMES

Recent studies with adeno-associated virus (AAV) have shown that site- specific integration is directed by DNA sequence motifs that are prese nt in both the viral replication origin and the chromosomal preintegra tion DNA and that specify binding and nicking sites for the viral regu latory Rep protein. This finding raised the question as to whether oth er parvovirus regulatory proteins might direct site-specific recombina tion with DNA targets that contain origin sequences functionally equiv alent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication o rigin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replic ates as an episome in a cell-cycle-dependent manner in mammalian cells . Upon MVM infection of these cells, the infecting genome integrated i nto episomes containing the active-origin sequence reported to be effi ciently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of th e origin sequence reported to be inefficiently nicked by NS1 or the ac tive form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several f eatures previously described for AAV episomal and chromosomal recombin ants. The findings indicate that the rules which govern AAV site-speci fic recombination also apply to MVM and suggest that site-specific chr omosomal insertions may be achievable with different autonomous parvov irus replicator proteins which recognize binding and nicking sites on the target DNA.