Project Abstract This project aims to use two highly novel and powerful assays to investigate HIV-1 protease (PR) precursor dimerization and autoprocessing in the context of intact viruses. In contrast to studies of the mature PR, little is known about how the precursor PR is activated and regulated, particularly under physiological conditions. We have developed a flow virometry-based assay that enables high-throughput analysis and sorting of individual viral particles and provides a rapid and efficient method for identifying determinants of precursor PR activation. To complement this method, we have also developed a selective reaction monitoring-mass spectrometry (SRM-MS) assay that allows simultaneous monitoring of all Gag and Gag-Pol cleavage sites with extremely accurate quantification of cleavage site processing efficiency. We will use these assays to: Aim 1: Identify determinants of precursor PR autoprocessing in the context of intact viruses. Aim 2: Determine the timing of PR activation in relation to budding, and define how premature activation of precursor PR activity is regulated. Aim 3: Evaluate how drug resistance and accessory mutations affect processing, budding, and the infectivity of viruses under physiological conditions.