Abstract
BackgroundCircular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription.ResultsWe demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette.ConclusionOur data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile.
Highlights
Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1
By including the Flp recombination target (FRT) sequence in this vector, we reasoned that Lentiviral vectors (LV) DNA circles generated during vector transduction would serve as substrates for Flp-dependent site-directed insertion of viral DNA into FRT sites engineered into the genome of transduced cells
Site-specific integration systems as Cre and Flp are important tools in genetic engineering and animal transgenesis but can in some instances be hampered by the requirement for transfection of plasmid DNA
Summary
Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are deadend products of reverse transcription. Great interest is attracted to ways of altering the lentiviral integration profile, allowing gene insertion in predetermined and safe vector landing sites. It remains unknown, whether lentiviral integration can be directed by alternative integration machineries, despite the integrity of the PIC and the exquisite involvement of cellular factors in nuclear entry and chromatin association
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