Project Summary The process of HIV-1 virion budding is essential for the completion of viral egress and intrinsically linked to host cellular machinery, yet the structural mechanisms by which budding occurs remain poorly understood. While the HIV-1 Gag polyprotein is capable of deforming host cells membranes and generate bud structures, HIV-1 must hijack the host ESCRT proteins, which ultimately drive the membrane scission event required for viral budding. This process begins with the recruitment of early-ESCRT factors (ESCRT-I, ESCRT- II, and ALIX) to the viral bud site by HIV-1 Gag. These early ESCRTs are then responsible for the recruitment of the late-ESCRT proteins, which form filamentous structures that are believed to drive scission through an active remodeling process. Despite growing amounts of structural, biochemical, and biophysical data on these proteins, the structural mechanism by which ESCRTs drive membrane scission is unknown, leaving a significant gap in our understanding of how HIV-1 release is accomplished. In Aim 1, I will determine the structures of 1:1 early-ESCRT complexes (ESCRT-I/ESCRT-II and ESCRT-I/ALIX) assembled on lipid membranes, using single-particle cryo-electron microscopy. Recent structural work from the Hurley lab has shown that ESCRT-I can self-associate to form spiral polymers through interactions within its core domain, implying that ESCRT-I's interactions with ESCRT-II or ALIX may serve as an early template for organizing the full ESCRT scission machinery. Results from this Aim will reveal the structures of these complexes in a native- like context, providing valuable information to how the early-ESCRT components assemble at the membrane and promote ESCRT-III recruitment. In Aim 2, a scission-competent, controllable in vitro viral budding system will be developed and structures of the full ESCRT machinery at these viral bud necks will be determined using cryo-electron tomography. The structures determined in this Aim will represent the first snapshots of on- pathway membrane scission by the ESCRT machinery. Such structures will not only reveal the molecular organization of the full ESCRT assembly, but also allow for the development, testing, and realization of the exact structural mechanism behind ESCRT-mediate membrane scission. Detailed cryo-EM and cryo-ET studies of these supramolecular complexes will reveal how HIV-1 hijacks and organizes the ESCRT machinery to complete a vital aspect of the viral life cycle. This basic research proposal is significant for future antiretroviral discovery, as our current incomplete mechanistic understanding of HIV-1 release has limited investigations into its potential as a target for antiretroviral therapies. This work will result in a detailed `movie' of—and biophysical insights into—the steps leading to virion budding and release. This contribution will be significant because it will reveal the extent to which antivirals might successfully target HIV-1 release, inclu...