Activation of the HIV-1 protease (PR) is an essential step in viral replication. Although clinically available substrate-based PR inhibitors have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. The utility of these agents is further limited by the phenomenon of cross-resistance between members of the same class. This problem appears to be especially acute for the agents that target the PR active site. Given the increasing prevalence of antiviral resistance, the somewhat limited efficacy of salvage regimens and the problems related to cross-resistance, novel approaches to inhibitor design are urgently needed. Although the available inhibitors of the HIV PR were developed using the 99 amino acid mature protease as a target, the PR is initially translated as part of the 160 kDa GagPol precursor. Processing of this precursor by the viral PR is a critical step in viral replication. As is the case for all retroviral PRs, HIV PR is only active as a homodimer and this embedded, immature PR is sufficient for precursor processing. The functional consequence of this arrangement is that the PR domains of two GagPol precursors must dimerize, become activated and begin to process the precursor in order for the viral life cycle to proceed. Mutations that interfere with the activation of the PR within GagPol have a profound effect on infectivity. We propose to develop an ELISA-based high throughput screen (HTS) for identifying compounds that inhibit the activation of the HIV PR embedded within the GagPol precursor. This system is based on our ability to express full length GagPol and monitor PR activation by measuring the release of an epitope tag. Specifically, we will: 1. Increase the sensitivity of our detection system for compounds that inhibit activity of the HIV PR embedded within GagPol. 2. Decrease the background activation of the GagPol PR.