VIRAL LONG TERMINAL REPEAT SUBSTRATE-BINDING CHARACTERISTICS OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhi bited a nonlinear dependence on integrase concentration, suggesting th at the enzyme functions as a multimer. The oligomeric state was invest igated by UV-photo-cross-linking of integrase-LTR oligonucleotide comp lexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine w ithin the integrase recognition sequence. In the absence of divalent c ation, integrase cross-linked to the LTR oligonucleotide as a single s pecies whose mobility by SDS-polyacrylamide gel electrophoresis was co nsistent with the formation of tetramers. Using these techniques, anal ysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzy me are distinct, involving residues within the conserved ''DD(35)E'' a nd zinc finger motifs, respectively.