BIOCHEMICAL-CHARACTERIZATION OF THE HIV-1 INTEGRASE 3'-PROCESSING ACTIVITY AND ITS INHIBITION BY PHOSPHOROTHIOATE OLIGONUCLEOTIDES

To better understand HIV-1 integrase (IN) functions, we determined the kinetic parameters of the 3'-processing reaction. Steady-state kineti c analysis performed using Dixon plots indicated that the concentratio n of active enzyme was 10-fold lower than that calculated by protein d etermination. The turnover number was low, suggesting that IN remained bound to DNA after cleavage. The catalytic efficiency increased 10-fo ld from 30 to 37 degrees C and 2-fold from 37 to 42 degrees C. In enzy me assays carried out at 37 degrees C, both single-and double-stranded phosphorothioate oligos bound to IN with an efficiency comparable to that of the phosphodiester duplex substrate. The competition efficienc y of single-stranded oligos was directly related to the sequence lengt h. On the other hand, phosphorothioate duplex U5 LTRs modified in the plus strand were capable of both competing with the substrate and dire ctly inhibiting the 3'-processing activity. These results suggest that , in addition to other modes of action (inhibition of gp120-CD4 intera ction and reverse transcriptase), phosphorothioate hetero- and homopol imeric oligos also potently inhibit the IN activity.