HIV replication involves reverse transcription of the viral RNA to make a cDNA copy, and integration of that cDNA into a chromosome of the host cell. Reverse transcriptase (RT) has been exploited as a target for antiviral agents, as has viral protease (PR), which is required for maturation of the viral proteins. The third HIV-encoded enzyme, integrase (IN), has not yet been exploited as an inhibitor target. We propose to carry out a thorough study of the chemistry of HIV-1 IN function, with the dual goal of elucidating the reaction mechanism and providing data to help guide the design of inhibitors. In our first series of studies, we propose to probe the IN active site using metal rescue experiments, mutagenic probing of catalytic requirements, and tethering of new small molecules in or near the active site. The second series of studies will focus on elucidating the structure of the integrase-DNA complex. There is now considerable evidence that IN protein correctly assembled with its DNA substrates responds differently to small molecule inhibitors than does free IN protein--thus the key issue in designing IN inhibitors is understanding the full IN-DNA complex. A major complication in studying IN-DNA complexes is the non-specific DNA binding by IN that predominates after mixing IN and DNA in solution. We have devised methods for assembling homogenous and monodisperse IN-DNA complexes based on use of 1) disulfide-mediated cross-linking of IN to DNA, and 2) assembly of IN with branched DNAs resembling integration intermediates. We propose to exploit these complexes and other experimental paradigms to characterize the chemistry of HIV-1 IN function using X-ray crystallography, NMR, cryo-electron microscopy, FRET and other methods. The Specific Aims are: Aim 1. Functional studies of IN-DNA complexes Aim 2. Structural analysis of IN-DNA complexes.