Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type I integrase and Escherichia coli LexA protein

We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein, A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed, The fusion protein, IN1-2 88/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragments as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein, No bias was observed when the LexA-binding sequence was absent, I hen the fusion protein was replaced by WT IN, or when Lexa protein was added in the reaction containing IN1-288/LA, A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence, The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase, The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.