Project Summary. Human Immunodeficiency Virus Type-1 (HIV-1) assembly mechanisms, namely the acquisition of the envelope glycoprotein (Env) into the assembling viral lattice, remains inadequately understood. The continued innovation of tools to interrogate these molecular and nanoscale assembly processes is beginning to disclose some of the basic principles. This proposal aims to build upon these innovations and quantify the requirements for Env assembly by interrogating viral and host factor determinants regulating intracellular trafficking, surface display, virus incorporation, and cell-to-cell spread. To accomplish these aims, we will adapt current superresolution and single-molecule tracking approaches developed by our laboratory to understand the three-dimensional structural organization and spatiotemporal dynamics of Env throughout assembly stages and cell-to-cell spread. Next, we will dissect the long Env cytoplasmic tail to understand which residues/regions drive Env incorporation. Finally, we propose to identify the role of host trafficking factors in regulating the surface levels of Env. Our preliminary data supports the feasibility of our approach by demonstrating our ability to extract quantitative information regarding the nanoscale organization and single-molecule dynamics of Env in three- dimensions. These collective approaches will shed new light on HIV-1 assembly and cell-to-cell virus transfer, informing new models and approaches aimed at targeting these processes therapeutically.