DIFFERENTIAL DIVALENT-CATION REQUIREMENTS UNCOUPLE THE ASSEMBLY AND CATALYTIC REACTIONS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to a ssemble as a stable complex on the donor substrate and to catalyze str and transfer. We now demonstrate that subsequent to assembly, catalysi s of both 3' end processing and strand transfer requires a divalent ca tion cofactor and that the divalent cation requirements for assembly a nd catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cati on requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end proce ssing and strand transfer assays are then used in conjunction with exo nuclease III protection analysis to investigate the effects of integra se inhibitors on each step in the reaction. Analysis of a series of re lated inhibitors demonstrates that these types of compounds affect ass embly and not either catalytic process, therefore reconciling the appa rent disparate results obtained for such inhibitors in assays using is olated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysi s and have implications for both the identification and characterizati on of integrase inhibitors.