Modulating Target Site Selection During Human Immunodeficiency Virus DNA Integration In Vitro with an Engineered Tethering Factor

The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage λ repressor protein (λR). In the presence of the λR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near λR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein–protein interactions might be engineered to constrain integration site selection during human gene therapy.