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 integrase (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 integration sites are selected is unknown, although it is clear that many regions are accessible to the retroviral integration machinery. To investigate integration site selection, and to possibly influence this process, we have used a model system in which the avian sarcoma virus (ASV) IN, 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 cognate lexA operators in vitro. Binding of precise N- and C-terminal fusion 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 experiments which revealed that the highly conserved "zinc-finger" domain of ASV IN is not essential for processing of viral DNA ends or joining of viral DNA ends to DNA target sequences. Last, the C-terminal fusion 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 heterologous domain. These observations have implications for retroviral-mediated gene therapy.
Katz
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RA
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Merkel
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Skalka
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AM
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002642:Chick Embryo; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D019426:Integrases; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D012697:Serine Endopeptidases; D019065:Virus Assembly; D016662:Virus Integration; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004254:DNA Nucleotidyltransferases; D004279:DNA, Viral; D019426:Integrases; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein