Analysis of integration activity of human immunodeficiency virus type-1 integrase

The integration activity of human immunodeficiency virus type-1 (HIV-1) int egrase was characterized in vitro by using pre-processed oligonucleotide su bstrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 7.0. Altho ugh the endonucleolytic and integration reactions are consecutive in retrov iral integration, our result indicates that the optimal conditions of the t wo reactions are quite different. In addition, it is suggested that the end onucleolytic and integration steps can be separated by control of the cellu lar physiological state in retroviral therapy Strong integration was detect ed in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion, This observation explains that the Mn2+ ion is preferred to t he Mg2+ ion as a cofactor in the integration reaction. Although there was n o sequence-specificity in the integration site of the target DNA, integrati on was found to frequently occur at particular regions of the target DNA, F urthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity comp letely, indicating that these residues are critically involved in catalytic action.