Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites.
In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.
Tan
Wenjie
W
Department of Molecular and Medical Pharmacology, Molecular Biology Institute, and UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
Zhu
Kai
K
Segal
David J
DJ
Barbas
Carlos F
CF
Chow
Samson A
SA
D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D016662:Virus Integration; D016335:Zinc Fingers / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019427:HIV Integrase