Abstract
The integration protein of the human immunodeficiency virus type 1 was purified from recombinant bacteria overproducing this enzyme. The final step of purification, namely chromatography on polyUsepharose, yielded a homogeneous protein preparation showing specific DNA cutting and joining activities. For a convenient assay of the endonuclease reaction, a 21-mer duplex oligonucleotide corresponding to the U5-LTR end of the viral DNA was radiolabeled at the dinucleotide that is removed by the enzyme. After the reaction, assay mixtures were passed through DEAE-cellulose filters which bind the substrate, but not the short radiolabeled product. Thus, an enzyme-dependent decrease of bound radioactivity was observed with time. Reaction rate was linearly dependent on enzyme concentration and the amount of substrate used was far below saturating concentrations. The reaction showed a pH-optimum at 7.5 and was strictly dependent on the presence of Mn2+. The presence of reducing agents like 2-mercaptoethanol was not essential for enzymatic activity. The assay was used to test selected compounds for their inhibitory potential against integrase. Typical inhibitors of DNA-topoisomerases did not inhibit the endonuclease reaction, with the exception of the intercalative agent actinomycin D which blocked the reaction with an IC50-value of 3 μM. Dextran sulphate inhibited the enzyme with an IC50 = 1.6 μg ml−1.