HIV-1 assembly is driven by the viral Gag polyprotein, with host factors contributing essential activities. We and others have shown that host machinery of the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway mediates the membrane fission reaction that releases HIV-1 virions (termed “budding”), that the HECT ubiquitin E3 ligase NEDD4L stimulates release of HIV-1 constructs that cannot recruit ESCRT factors directly, and that members of the Angiomotin (AMOT) family of NEDD4L-binding proteins promote progression of the assembling Gag lattices from hemispheres to membrane-enveloped spheres (termed “envelopment”). Most recently, we have discovered that multiple different mammals have independently evolved truncated, retrotransposed copies of a CHMP3 (ESCRT-III) protein that can potently inhibit release of HIV-1 and other ESCRT-dependent enveloped viruses without undue cellular toxicity. We now propose to build on these observations by pursuing complementary structural, biochemical, imaging, and functional approaches to address three central questions in HIV-1 biogenesis: 1) How do assembling virions become wrapped in membranes? 2) How does the ESCRT machinery catalyze viral membrane constriction and budding? 3) How can mammals protect themselves broadly against ESCRT-dependent viruses? In addition to their relevance for HIV, each of these processes has analogs in other viral and/or cellular systems, which should extend the impact of our studies. Specifically, we propose to characterize how AMOT-NEDD4L complexes contribute to HIV virion envelopment (Aim 1), how late-acting ESCRT-III filaments and VPS4 ATPases collaborate to constrict membranes and promote fission (Aim 2), and how truncated, retrotransposed CHMP3 proteins (retroCHMP3) can inhibit retroviral budding without inducing cellular toxicity (Aim 3). These Aims are buttressed by our structural studies showing how the AMOT PPXY1 and NEDD4L WW3 domains form a high affinity complex (Aim 1), how ESCRT-III proteins form soluble, monomeric proteins and membrane- bound filaments (Aims 2 and 3), and how VPS4 ATPases bind these filaments and remove ESCRT-III subunits (Aim 2). Each Aim will also be supported by biochemical assays designed to elucidate how the AMOT-NEDD4L complex remodels membranes, stabilizes F-actin, and activates NEDD4L ubiquitin E3 ligase activity (Aim 1), how essential ESCRT-III subunits co- assemble and constrict membranes (Aim 2), and how retroCHMP3 interferes with ESCRT-III filament formation at sites of virus budding and whether these proteins function as restriction factors in their natural settings (Aim 3). All of the Aims will be facilitated by genetic and imaging assays that will help reveal the activities of AMOT, NEDD4L, ESCRT-III, VPS4 and retroCHMP3 variants as they function at cellular sites of HIV-1 assembly and budding. Our goal is to use these complementary approaches to generate and test mechanistic models for three fundamental processes in HIV-1 bi...