Cloning and sequencing of defective particles derived from the autonomous parvovirus minute virus of mice for the construction of vectors with minimal cis-acting sequences

The production of wild-type-free stocks of recombinant parvovirus minute vi rus of mice [MVM(p)] is difficult due to the presence of homologous sequenc es in vector and helper genomes that cannot easily be eliminated from the o verlapping coding sequences. We have therefore cloned and sequenced spontan eously occurring defective particles of MVM(p) with very small genomes to i dentify the minimal cis acting sequences required for DNA amplification and virus production. One of them has lost all capsid coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with n ew, matched, helper plasmids that present no homolog downstream from the tr ansgene.