PROJECT SUMMARY Studies in this Project are undertaken with the goal of advancing foundations required to tackle new frontiers in HIV-1 biology and medicine, including the development of cure strategies, broad antiviral therapeutics, and methods for delivery of biologic therapeutics into target cells. To these ends, studies in Project 3, Multiscale Analysis and Modulation of Viral Dynamics, will characterize HIV-1 proviral silencing and reactivation over a wide range of resolution scales, from living animals to high-resolution structural studies, and will develop new methods aimed at protecting animals from enveloped viral infections and designing new biologics delivery systems. Studies in Aim 1 (In Situ Architecture of Virus Reactivation) will examine, across multiple size and resolution scales, viruses and associated cells at sites of HIV/SIV rebound in animal models. Specifically, we will: 1) locate and image reactivating virus and associated cells, and 2) define the transcription profiles of infected and neighboring cells at rebound sites. Studies in Aim 2 (Proviral Silencing and Reactivation) seek a molecular and structural understanding of how HIV-1 silencing is established and maintained. To this end, we will determine structures, interactions, and mechanisms important for latency, with a focus on the H3K9-specific methylase SETDB1, and on HUSH, which localizes on H3K9me3 and collaborates with SETDB1 to promote the spread of H3K9me3 and heterochromatin. Studies in Aim 3 (RetroCHMP3 Blocks to Viral Dissemination) will build on our recent discovery of retroCHMP3 proteins, which are naturally-occurring factors that potently inhibit release of enveloped viruses that use the ESCRT pathway for budding, including HIV-1. Our goals are to optimize retroCHMP3 potency in mouse cells lines by increasing protein stability, expression, and restriction activity, and then create transgenic mice that express the optimal constructs and test for broad resistance to enveloped viruses. Studies in Aim 4 (Virus-Inspired Designed Delivery Systems) will generate new virus-inspired technology platforms for intercellular delivery that are simple, robust, and controllable by taking advantage of: 1) recent developments in computational protein design, 2) our previous successes in harnessing the principles of HIV-1 assembly to develop nanoparticles that can direct their own self-assembly and bud from cells, and 3) our new advances for incorporating transmembrane proteins into those particles.