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) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.
Harper
A L
AL
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
Skinner
L M
LM
Sudol
M
M
Katzman
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015658:HIV Infections / Q000821:virology*; D019427:HIV Integrase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D016662:Virus Integration
D004247:DNA; D019427:HIV Integrase