TARGETING OF RETROVIRAL INTEGRASE BY FUSION TO A HETEROLOGOUS DNA-BINDING DOMAIN - IN-VITRO ACTIVITIES AND INCORPORATION OF A FUSION PROTEIN INTO VIRAL PARTICLES

Retroviral DNA integration is catalyzed by a viral protein, the integr ase (IN). IN recognizes sequences at the viral DNA ends, specifically nicks these ends (the processing reaction), and inserts them into host DNA (the joining reaction). The mechanism by which host DNA integrati on sites are selected is unknown, although it is clear that many regio ns are accessible to the retroviral integration machinery. To investig ate integration site selection, and to possibly influence this process , we have used a model system in which the avian sarcoma virus (ASV) I N, and segments thereof, have been fused to the Escherichia coli LexA repressor protein DNA binding domain (DBD). We provide evidence that a variety of such IN-LexA DBD fusion proteins can be directed to cognat e lexA operators in vitro. Binding of precise N- and C-terminal fusion s proteins to the operators is associated with a significant increase in the joining of viral DNA to sites immediately adjacent to the lexA operator region. We also carried out domain exchange and deletion expe riments which revealed that the highly conserved ''zinc-finger'' domai n of ASV IN is not essential for processing of viral DNA ends or joini ng of viral DNA ends to DNA target sequences. Last, the C-terminal fus ion was engineered into the viral genome and we show that this protein can be incorporated into viral particles. Our results suggest that it might be possible to augment IN function in vivo through a heterologo us domain. These observations have implications for retroviral-mediate d gene therapy. (C) 1996 Academic Press, Inc.