Project Summary: Our long-term goal is to gain a complete understanding of the molecular machinery that regulates the release of infectious HIV virions from infected cells. During our previous funding period (R21), we found that early Endosomal Sorting Complexes Required for Transport play a major role in a molecular race between virion budding and Gag-Pol auto-processing. We also found that disturbing the balance between budding and auto- processing results in release of non-infectious virions. In the face of rapid evolution of HIV under protease inhibitors, search for new target mechanisms to curb infections is crucial. Gag-Pol auto-processing and its regulation during assembly and budding present an intriguing target. Our fundamental understanding of this process however is limited due to its entanglement with assembly and budding kinetics and asynchronous nature of virion release. Here we propose to visualize HIV budding and Gag-Pol auto-processing in single virions in vivo to: 1) Establish the dynamics and regulation of early ESCRT recruitment during full HIV virion assembly, 2) Measure the dynamics of Gag-Pol incorporation and auto-processing during HIV budding and 3) Dissect the regulation of Gag-Pol auto-processing.