Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells.
Department of Molecular and Medical Pharmacology, Molecular Biology Institute, and UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA.
In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.
PMID: 16439549 [PubMed - indexed for MEDLINE]
PMCID: PMC1367172