Project Summary The human immunodeficiency virus type 1 (HIV-1) is a retrovirus that completes its viral life cycle when the newly assembled virion is released from the infected cell. The HIV-1 structural protein, Gag, recruits the host cell’s endosomal sorting complex required for transport (ESCRT) proteins towards the neck of budding virions to sever the nascent viral particle from the plasma membrane. In addition to viral-like particle (VLP) release, ESCRTs are utilized in membrane scission of other topologically equivalent cellular processes such as vesicular trafficking, cell division, exosome biogenesis, and plasma membrane repair. Recent advances in molecular biology and virology highlight the importance and timing of recruitment of ESCRTs in achieving proper viral particle release. However, a description of the biophysical mechanism of ESCRT-mediated scission during HIV release remains a major goal in the field. Newly developed methods in our laboratory have now made possible the encapsulation of functional human ESCRT and Gag proteins inside giant unilamellar vesicles (GUVs), thereby recapitulating the correct topology for ESCRT function in vitro. We have integrated a high-speed confocal microscope with optical trapping capabilities which allows the visualization and investigation, with piconewton resolution, of the force generated during scission of a single membrane neck. Control of the reaction is modulated by UV photolysis of a caged-nucleotide for this ATP-dependent process. Together, these innovations have given us a unique ability to interrogate HIV-1 release by ESCRTs under a biophysical lens. This proposed research represents a focused and innovative approach to investigate the ATP-dependent membrane scission mechanism of human ESCRT proteins in the setting of HIV-1 release; directly building and expanding on our previous success with the yeast ESCRT system. Specifically, in aim 1, I will identify the scission mechanism of human ESCRTs. Subsequently, aim 2 will provide a biophysical explanation of the interplay between Gag binding and membrane scission by the ESCRT machinery. The overarching hypothesis for this proposal is that ESCRTs apply mechanical force to membrane necks which destabilizes them and leads to their scission. Ultimately, successful completion of this work allows for a detailed biophysical understanding of HIV-1 release by ESCRTs that may lead to the design of novel antivirals.