Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integ rases for alterations in the choice of nonviral target DNA sites. This stra tegy took advantage of the genetic diversity, of HIV-1, which provided 75 i ntegrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites w ould be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequen ces of proteins with the same preferences then guided mutagenesis of a labo ratory integrase. The results showed that single amino acid substitutions a t one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results we re found with DNA joining reactions conducted with Mn2+ or with Mg2+ and we re confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel a pproach has identified a residue in the central domain of HIV-1 integrase t hat interacts with or influences interactions with cellular DNA. The data a lso support a model in which integrase has distinct sites for viral and cel lular DNA.