The early intracellular events in human immunodeficiency virus (HIV) infection, leading to the establishment of the integrated provirus, remain inadequately understood. We hypothesize that many steps in this process, presently unrecognized or unexplored, could provide new targets against which antiviral drugs might be developed. One of the better understood steps in early infection is integration. Integration has clearly been shown to be required for HIV replication, and the biochemistry of integration is understood in broad outline. Yet we are still far from a detailed understanding of integration, and consequently the current approach to developing drugs targeted at integrase may be naive. Indeed, consideration of the place integration occupies in the sequence of events leading to integration in vivo suggests that the most straightforward approach to blocking integration, based on inhibitors of the catalytic activities of integrase, may not be the most effective strategy for preventing its occurrence in an actual infection. The goal of the proposed work is simply to explore and develop the most promising avenues toward discovery of new antiviral agents directed in integration and other early steps in the infection process. Four projects are planned: 1. Continued basic investigations of the biochemistry of integration, aimed at developing a complete description of the regulation, specificity, biochemical mechanism and structure of integrase, and other possible actors in the process. As an integral part of this basic work we will continue to develop assays to test each activity that we can isolate, for possible use in primary or secondary screens of candidate drugs. 2. Exploring new strategies for antiviral agents directed at integrase and testing their feasibility by using mutations in integrase as surrogates for inhibitors. 3. Uncovering new targets for antiviral agents in early infection, using two genetic strategies. We will use a powerful new genetic approach to screen for new mutations in the gag and pol coding regions that impair specific steps in early infection. We will also continue work in progress, using the yeast two-hybrid system to screen for cellular proteins that interact with the Gag or Pol proteins of HIV or murine leukemia virus (MLV). 4. We will define the mechanism of action of integrase inhibitors identified by the ongoing random screens being conducted by the Parke-Davis group.
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