PROJECT SUMMARY Tripartite motif (TRIM) proteins are ubiquitin E3 ligases that function in development and innate immunity. TRIM5a and TRIMCyp proteins (collectively termed TRIM5) recognize and deactivate the incoming cores of retroviruses such as HIV- 1. Indeed, human TRIM5a is an important component of interferon-mediated control of HIV-1 infection. Previous work from our group suggested an unprecedented lattice-to-lattice mode of pattern recognition, in which TRIM5a dimers use the viral capsid surrounding the viral core as a template for higher-order assembly, ultimately forming a cage around the core. In the current funding period of this R01 project, we solved the first structure of the TRIM5a/capsid superlattice, developed and optimized a recombinant assembly and ubiquitination system for TRIM5/capsid complexes, and collaborated in studies that reconstituted the early events of HIV-1 replication in a cell-free system. In this renewal application, we now propose to: Aim 1, extend the resolution of our TRIM5a/capsid superlattice structure, in order to visualize contacts between the arrayed TRIM5a protein dimers and their bound viral CA protein hexamer subunits. Aim 2, utilize our novel in vitro ubiquitination and HIV-1 replication systems as experimental platforms to elucidate how TRIM5 deactivates its caged viral core. Aim 3, understand a novel mechanism of TRIM5 recognition, in which the homologous TRIM34 protein directs human TRIM5a to bind and inactive HIV-1 capsids bearing the N74D mutation. Collectively, these studies will fill in important missing gaps in our understanding of how TRIM5 proteins inhibit HIV-1 replication. Our developing understanding of TRIM5 has been giving important insights on HIV-1 capsid vulnerabilities and efforts to develop drugs that exploit these vulnerabilities.