PROJECT SUMMARY (See instructions): The assembly and release of HIV-1 from infected cells are essential steps in the viral replication cycle. HIV assembly is driven by the virally encoded Gag polyprotein. Bending of the plasma membrane into spherical buds, packing of the RNA genome (gRNA), and incorporation of the envelope glycoprotein (Env) are among the key events of assembly and budding. Release depends on the the host-encoded ESCRT proteins, which are recruited by Gag to the neck. Building on considerable progress in understanding structural and cellular mechanisms of HIV assembly, we are now in a position to answer the major open questions about how HIV-1 orchestrates its own release by hijacking ESCRT complexes. This process is especially timely in the wake of the discovery of the endogenous retroCHMP3 factor that restricts ESCRT-mediated release of retroviruses and other enveloped viruses in some new world monkeys, without unduly compromising host ESCRT functions needed for normal cell function. We will take a three-pronged approach to explaining how HIV-1 assembly and release are coupled through the ESCRT system. In the first aim, structural studies of biochemically tractable subassemblies of ESCRTs from humans and other species will be used to build up experimentally validated models of those aspects of the system that are still missing. These include the linkages downstream of ESCRT-1. Single particle cryo-EM structures will be determined and integrated models will be build in collaboration with Greg Voth. In the second aim, the entire system consisting of HIV-1 Gag, membranes, and human ESCRTs will be reconstituted in vitro. Scission function will be assessed biophysically using membrane nanotubes pulled by optical tweezers. Structures of the entire reconstituted system will be determined by cryo-electron tomography (cryo-ET) and subtomogram averaging {STA), with modeling guided by atomistic structures, either pre-existing or obtained in the first aim. In the third aim, a structural movie of release will be obtained by stepwise in situ cryo-ET imaging of HIV-1 as it escapes infected cells. ESCRT-mediated HIV-1 release will be trapped at various stages through the use of targeted inhibition of each step in the assembly. We will begin with targeted inhibition of the AAA+ ATPase responsible for the final stage release, VPS4A/B. RetroCHMP3 and other targeted dominant negative factors will be used to inhibit earlier steps in the pathway. Cryo-ET imaging will be carried out for budding events at the cell periphery,