Control of parvovirus DNA replication by a tetracycline-regulated repressor

Autonomous parvoviruses are small, single strand DNA viruses which preferen tially replicate in transformed and tumor cells, causing cell death by expr ession of the cyto-toxic nonstructural protein, NS1. Several parvoviruses o f the rodent group, including LuIII, efficiently infect human transformed c ell lines. The potential for systemic use of These viruses in targeting met astases might be enhanced if NS1 expression and viral replication could be controlled by an innocuous drug such as tetracycline. We therefore substitu ted prokaryotic tetracycline operator sequences for part of P4 of LuIII, th e promoter responsible for transcription of the mRNAs for nonstructural pro teins. The resulting construct unexpectedly showed constitutive expression in transiently transfected cells, as indicated by efficient excision and am plification of viral replicative form (RF) DNA. This was apparently due to self-stimulatory transcriptional transactivation by NS1. This problem was o vercome by cotransfection with a plasmid expressing a chimera of the repres sor of the tetracycline operon with a KRAB trans-repression domain. These c onditions allowed efficient control of transcription and RF amplification b y the tetracycline derivative, doxycycline. These observations form a basis for developing a therapeutic agent based on a drug-controlled parvovirus.